Closed-ended linear duplex dna for non-viral gene transfer

ABSTRACT

Aspects of the disclosure relate to a nucleic acid comprising a heterologous nucleic acid insert flanked by interrupted self-complementary sequences, wherein one self-complementary sequence is interrupted by a cross-arm sequence forming two opposing, lengthwise-symmetric stem-loops, and wherein the other of the self-complementary sequences is interrupted by a truncated cross-arm sequence. Methods of delivering the nucleic acid to a cell are also provided.

RELATED APPLICATIONS

This application is a National Stage Application of PCT/US2017/020828,filed Mar. 3, 2017, entitled “CLOSED-ENDED LINEAR DUPLEX DNA FORNON-VIRAL GENE TRANSFER”, which claims the benefit under 35 U.S.C.119(e) U.S. Provisional Application 62/303,047, filed on Mar. 3, 2016,entitled “CLOSED-ENDED LINEAR DUPLEX DNA FOR NON-VIRAL GENE TRANSFER”,U.S. Provisional Application 62/394,720, filed Sep. 14, 2016, entitled“CLOSED-ENDED LINEAR DUPLEX DNA FOR NON-VIRAL GENE TRANSFER”, and U.S.Provisional Application 62/406,913, filed Oct. 11, 2016, entitled“CLOSED-ENDED LINEAR DUPLEX DNA FOR NON-VIRAL GENE TRANSFER”, the entirecontents of each application which are incorporated herein by reference.

BACKGROUND

Current gene delivery vectors have several drawbacks. Both viral andbacterial-derived gene delivery vectors can induce the innate andadaptive immune responses of a patient. For example, plasmid DNA (pDNA)and mini-circle DNA (mcDNA) vectors, typically have prokaryotic patternsof DNA methylation that are not present in eukaryotic DNA. Additionally,lipopolysaccharides (LPS) and other bacterial-derived molecules arerecognized in vertebrate cells by the innate immune response patternrecognition receptor (PRR) as pathogen-associated molecular patterns(PAMPs), leading to activation of cellular genes in response to theinvasive microbial pathogen. Plasmid DNA conformationally is uniquelybacterial; the closest mammalian structure is the mitochondrial genome,or duplex circular DNA, which compartmentalized in the organelle, is notexposed to the cytosolic PRRs. In another example, recombinantadeno-associated viruses (rAAVs) can induce a T-cell response toprocessed capsid antigens or be neutralized by circulatingimmunoglobulins and non-Ig glycoproteins. Viral vectors also havelimited transgene carrying capacity and are labor intensive, expensive,and time consuming to produce. Accordingly, improved compositions andmethods for gene delivery are needed.

SUMMARY

The disclosure relates, in some aspects, to the discovery thatreplication of nucleic acids encoding a heterologous nucleic acid insertflanked by certain types of asymmetric termini (e.g., asymmetricinterrupted self-complementary sequences) results in covalent linkage ofthe asymmetric termini (e.g., asymmetric interrupted self-complementarysequences) and leads to the production of a novel conformation ofclosed-ended linear duplex DNA (ceDNA). In some embodiments, nucleicacids having asymmetric interrupted self-complementary sequences can bereadily produced (e.g., in large quantities) while avoiding scale upissues associated with other gene therapy vectors (e.g., viral basedvectors). This result is surprising in view of reports that symmetry isrequired in the internal palindromic region for purposes of propagationof similar nucleic acids.

In some embodiments, nucleic acids having asymmetric interruptedself-complementary sequences, as disclosed herein, may have improvedgenetic stability compared with other gene therapy vectors (e.g.,nucleic acids having symmetric interrupted self-complementarysequences). In some embodiments, nucleic acids having asymmetricinterrupted self-complementary sequences, as disclosed herein, may haveimproved safety profiles compared with other vectors (e.g., nucleicacids having symmetric interrupted self-complementary sequences). Forexample, in some embodiments, administration of nucleic acids havingasymmetric interrupted self-complementary sequences may be less likelyto result in insertional mutagenesis compared with other vectors (e.g.,nucleic acids having symmetric interrupted self-complementary sequences)due to the asymmetric nature of the construct.

In certain embodiments, nucleic acids having asymmetric interruptedself-complementary sequences that are engineered to express a transcript(e.g., a transcript encoding a protein or functional nucleic acid) mayhave improved expression compared with other vectors (e.g., nucleicacids having symmetric interrupted self-complementary sequences) becausethe asymmetric nature of the constructs makes them less likely tointeract in cells with certain enzymes (e.g., helicases, such as, RecQhelicases) that can reduce the transcriptional capacity of such vectors.

In some embodiments, administration of a nucleic acid having asymmetricinterrupted self-complementary sequences, as described herein, is lesslikely to induce an immune response in a subject compared withadministration of other gene therapy vectors (e.g., plasmid DNA vectorsand viral vectors). Therefore, in some embodiments, a nucleic aciddescribed herein can be administered to a subject on multiple occasions(e.g., in the context of long-term gene therapy) without inducing asubstantial immune response that would prevent or inhibit expressionand/or activity of a gene product encoded by the nucleic acid.

In some aspects, the disclosure provides a nucleic acid comprising aheterologous nucleic acid insert flanked by at least one interruptedself-complementary sequence, each self-complementary sequence having anoperative terminal resolution site and a rolling circle replicationprotein binding element, wherein the self-complementary sequence isinterrupted by a cross-arm sequence forming two opposing,lengthwise-symmetric stem-loops, each of the opposinglengthwise-symmetric stem-loops having a stem portion in the range of 5to 15 base pairs in length and a loop portion having 2 to 5 unpaireddeoxyribonucleotides.

In some embodiments, interrupted self-complementary sequences arederived from a one or more organisms or viral serotypes, including fromparvoviruses, dependovirus, etc. For example, in some embodiments, anucleic acid comprises a first interrupted self-complementary sequencederived from an AAV2 serotype and a second interruptedself-complementary sequence derived from an AAV9 serotype. In anothernon-limiting example, a nucleic acid as described by the disclosure maycomprise a first interrupted self-complementary sequence from an AAV2serotype and a second interrupted self-complementary sequence from aparvovirus (e.g., parvovirus B19). In some embodiments, interruptedself-complementary sequences are derived from the same organism or viralserotype but have different lengths, or combinations of the foregoing.In some embodiments, the nucleic acid comprises a second interruptedself-complementary sequence that is interrupted by a truncated cross-armsequence. For example, in some embodiments, a nucleic acid comprises afirst self-interrupted self-complementary sequence derived from an AAV2serotype that is 145 base pairs in length, and a second interruptedself-complementary sequence derived from an AAV2 serotype that isshorter than 145 base pairs in length (e.g., a truncated cross-armsequence).

In some aspects, the disclosure provides a nucleic acid comprising aheterologous nucleic acid insert flanked by interruptedself-complementary sequences, each self-complementary sequence having anoperative terminal resolution site and a rolling circle replicationprotein binding element, wherein one self-complementary sequence isinterrupted by a cross-arm sequence forming two opposing,lengthwise-symmetric stem-loops, each of the opposinglengthwise-symmetric stem-loops having a stem portion in the range of 5to 15 base pairs in length and a loop portion having 2 to 5 unpaireddeoxyribonucleotides, wherein the other of the self-complementarysequences is interrupted by a truncated cross-arm sequence.

In some embodiments, the interrupted self-complementary sequence(s) arein the range of 40 to 1000 nucleotides in length. In some embodiments,the interrupted self-complementary sequence(s) are in the range of 100to 160 nucleotides in length.

In some embodiments, the cross-arm sequence has a Gibbs free energy (ΔG)of unfolding under physiological conditions in the range of −12 kcal/molto −30 kcal/mol. In some embodiments, the cross-arm sequence has a Gibbsfree energy (ΔG) of unfolding under physiological conditions in therange of −20 kcal/mol to −25 kcal/mol.

In some embodiments, each of the opposing lengthwise-symmetricstem-loops have a stem portion in the range of 3 to 15 base pairs inlength. In some embodiments, each of the opposing lengthwise-symmetricstem-loops have a stem portion in the range of 8 to 10 base pairs inlength.

In some embodiments, each loop portion has 2 to 5 unpaireddeoxyribonucleotides. In some embodiments, each loop portion has threedeoxyribonucleotides.

In some embodiments, one loop portion has three deoxythymidines and theother loop portion has three deoxyadenosines.

In some embodiments, the rolling circle replication protein bindingelement is a Rep binding element (RBE). In some embodiments, the RBEcomprises the sequence 5′-GCTCGCTCGCTC-3′ (SEQ ID NO: 1).

In some embodiments, the operative terminal resolution site comprises asequence 5′-TT-3′. In some embodiments, the 3′ end of the operativeterminal resolution site is 15 to 25 nucleotides from the 5′ end of therolling circle replication protein binding element.

In some embodiments, the truncated cross-arm sequence forms twoopposing, lengthwise-asymmetric stem-loops. In some embodiments, one ofthe opposing, lengthwise-asymmetric stem-loops has a stem portion in therange of 8 to 10 base pairs in length and a loop portion having 2 to 5unpaired deoxyribonucleotides. In some embodiments, the onelengthwise-asymmetric stem-loop has a stem portion less than 8 basepairs in length and a loop portion having 2 to 5 deoxyribonucleotides.In some embodiments, the one lengthwise-asymmetric stem-loop has a stemportion less than 3 base pairs in length. In some embodiments, the onelengthwise-asymmetric stem-loops has a loop portion having 3 or fewerdeoxyribonucleotides. In some embodiments, the truncated cross-armsequence has a Gibbs free energy (ΔG) of unfolding under physiologicalconditions in the range of 0 kcal/mol to −22 kcal/mol.

In some embodiments, the heterologous nucleic acid insert is engineeredto express a protein or functional RNA. In some embodiments, theheterologous nucleic acid insert is a promoterless construct as asubstrate for gene editing. In some embodiments, the promoterlessconstruct provides a substrate for TALENS, zinc finger nucleases (ZFNs),meganucleases, Cas9, and other gene editing proteins. In someembodiments, the promoterless construct is flanked by nucleic acid withhomology to cell DNA to promote homologous recombination into the cellgenome. In some embodiments, the construct is flanked by nucleic acidwith homology to cell DNA to promote homologous recombination into thecell genome.

In some embodiments, the nucleic acid is in the range of 500 to 50,000nucleotides in length. In some embodiments, the nucleic acid is in therange of 500 to 10,000 nucleotides in length. In some embodiments, thenucleic acid is in the range of 1000 to 10,000 nucleotides in length. Insome embodiments, the nucleic acid is in the range of 500 to 5,000nucleotides in length.

In some aspects, the disclosure provides a composition comprising aplurality of nucleic acids as described by the disclosure. In someembodiments, the plurality of nucleic acids is linked end-to-end. Insome aspects, the disclosure provides a composition comprising a nucleicacid as described by the disclosure and a pharmaceutically acceptablecarrier.

In some aspects, the disclosure provides a composition comprising: amonomeric nucleic acid comprising a single subunit; and, at least onemultimeric nucleic acid comprising two or more subunits, wherein eachsubunit of the monomeric nucleic acid and of the at least one multimericnucleic acid comprises a heterologous nucleic acid insert flanked byinterrupted self-complementary sequences, each self-complementarysequence having an operative terminal resolution site and a rollingcircle replication protein binding element, wherein oneself-complementary sequence is interrupted by a cross-arm sequenceforming two opposing, lengthwise-symmetric stem-loops and the other ofthe self-complementary sequences is interrupted by a truncated cross-armsequence. In some embodiments, each multimer has at least one, and insome cases only one, self-complementary terminal palindrome.

In some embodiments, the at least one multimeric nucleic acid is acomprises two subunits. In some embodiments, multimeric nucleic acid hasno more than two subunits. In some embodiments, the two subunits arelinked in a tail-to-tail configuration, or head-to-head configuration,or a head-to-tail configuration.

In some aspects, the disclosure provides host cell comprising thenucleic acid as described by the disclosure. In some embodiments, thehost cell further comprises a rolling circle replication protein thatselectively binds to the rolling circle replication protein bindingelement of the nucleic acid.

In some embodiments, the disclosure provides a method of delivering aheterologous nucleic acid to a cell, the method comprising delivering tothe cell a nucleic acid as described by the disclosure.

In some aspects, the disclosure provides a method of delivering aheterologous nucleic acid to a subject, the method comprising deliveringto the subject a nucleic acid as described by the disclosure, whereinthe delivery of the nucleic acid does not result in eliciting anacquired immune response against the nucleic acid in the subject. Insome embodiments, the immune response is a humoral response. In someembodiments, the immune response is a cellular response.

In some embodiments, the heterologous nucleic acid is delivered onmultiple occasions to the subject. In some embodiments, the number ofoccasions in which heterologous nucleic acid is delivered (e.g.,administered) to the subject is in a range of 2 to 10 times. In someembodiments, the number of occasions in which heterologous nucleic acidis delivered to the subject is hourly, daily weekly, biweekly, monthly,quarterly, semi-annually, or annually. In some embodiments, the numberof occasions in which heterologous nucleic acid is delivered to thesubject is the number of occasions as required to maintain a clinical(e.g., therapeutic) benefit.

In some aspects the disclosure provides a method of delivering aheterologous nucleic acid to a subject, the method comprising deliveringa host cell as described by the disclosure to the subject. In someembodiments, the host cell is a blood cell. In some embodiments, thehost cell is a progenitor (e.g., hematopoietic stem cell, HSC), myeloid,or lymphoid cell. In some embodiments, the host cell is delivered onmultiple occasions. In some embodiments, the frequency at which the hostcell is delivered on multiple occasions is determined by the half-lifeof the host cell. In some embodiments, the host cell is delivered onmultiple occasions to achieve (e.g., achieve and maintain) therapeuticbenefit.

In some aspects, the disclosure provides a method of preparing nucleicacids, the method comprising: (i) introducing into a permissive cell anucleic acid encoding a heterologous nucleic acid insert flanked by atleast one interrupted self-complementary sequence, eachself-complementary sequence having an operative terminal resolution siteand a rolling circle replication protein binding element, wherein theself-complementary sequence is interrupted by a cross-arm sequenceforming two opposing, lengthwise-symmetric stem-loops, each of theopposing lengthwise-symmetric stem-loops having a stem portion in therange of 5 to 15 base pairs in length and a loop portion having 2 to 5unpaired deoxyribonucleotides; and, (ii) maintaining the permissive cellunder conditions in which a rolling circle replication protein in thepermissive cell initiates production of multiple copies of the nucleicacid.

In some embodiments, the method further comprises the step of purifyingthe multiple copies of the nucleic acid. In some embodiments, thepurification comprises contacting the nucleic acid with a silica gelresin.

In some embodiments, the rolling circle replication protein is selectedfrom the group consisting of wild-type AAV Rep 78, AAV Rep 52, AAVRep68, and AAV Rep 40. In some embodiments, the set of rolling circlereplication proteins include at least one from AAV Rep 78 and AAV Rep68, and one from AAV Rep 52 and AAV Rep 40. In some embodiments, rollingcircle replication proteins are functionally equivalent derivatives ofwild-type AAV Rep proteins including truncated proteins or fusionproteins.

In some embodiments, the permissive cell is not a mammalian cell. Insome embodiments, the permissive cell is an insect or otherinvertebrate-species cell line, yeast cell line, or bacterial cell line.In some embodiments, the permissive cell is used for production, e.g.,Spodoptera frugiperda larva. In some embodiments, occluded recombinantAutograph californica multiple nucleopolyhedrosis virus (AcMNPV) havebeen used to infect S. frugiperda larvae for recombinant proteinproduction, for example via a current good manufacturing practice (cGMP)process for protein production in larvae.

In some embodiments, the rolling circle replication protein is encodedby a helper virus vector, optionally wherein the helper virus vector isAutograph californica multiple nucleopolyhedrosis virus (AcMNPV) vectoror a baculovirus expression vectors (BEV).

In some aspects, the disclosure provides a method of preparing nucleicacids, the method comprising: introducing into a permissive cell anucleic acid comprising a heterologous nucleic acid insert flanked byinterrupted self-complementary sequences, each self-complementarysequence having an operative terminal resolution site and a rollingcircle replication protein binding element, wherein oneself-complementary sequence is interrupted by a cross-arm sequence thatforms two opposing, lengthwise-symmetric stem-loops, wherein the otherof the self-complementary sequences is interrupted by a truncatedcross-arm sequence, wherein the permissive cell expresses a rollingcircle replication protein, but does not express viral capsid proteinscapable of packaging replicative copies of the nucleic acid into a viralparticle; and maintaining the permissive cell under conditions in whichthe rolling circle replication protein in the permissive cell replicatesthe nucleic acid.

In some aspects, the disclosure provides a method of preparing nucleicacids, the method comprising: introducing into a permissive cell anucleic acid comprising a heterologous nucleic acid insert flanked byinterrupted self-complementary sequences, each self-complementarysequence having an operative terminal resolution site and a rollingcircle replication protein binding element, wherein oneself-complementary sequence has been determined to be interrupted by across-arm sequence that forms two opposing, lengthwise-symmetricstem-loops, wherein the other of the self-complementary sequences hasbeen determined to be interrupted by a truncated cross-arm sequence,wherein the permissive cell expresses a rolling circle replicationprotein, but does not express viral capsid proteins capable of packagingreplicative copies of the nucleic acid into a viral particle; andmaintaining the permissive cell under conditions in which the rollingcircle replication protein in the permissive cell replicates the nucleicacid.

In some embodiments, the method further comprises isolating thereplicated nucleic acid from the permissive cell.

In some aspects, the disclosure provides a method of analyzing a nucleicacid, the method comprising: obtaining a nucleic acid preparationcomprising nucleic acid replication products isolated from a permissivecell, wherein the permissive cell comprises a nucleic acid comprising aheterologous nucleic acid insert flanked by interruptedself-complementary sequences, each self-complementary sequence having anoperative terminal resolution site and a rolling circle replicationprotein binding element, wherein one self-complementary sequence isinterrupted by a cross-arm sequence that forms two opposing,lengthwise-symmetric stem-loops, wherein the other of theself-complementary sequences is interrupted by a truncated cross-armsequence, wherein the permissive cell expresses a rolling circlereplication protein, but does not express viral capsid proteins capableof packaging replicative copies of the nucleic acid into a viralparticle, and wherein the rolling circle replication protein binds tothe rolling circle replication protein binding element of the nucleicacid and replicates the nucleic acid to produce nucleic acid replicationproducts; and determining a physiochemical property of one or morereplication products.

In some embodiments, the physiochemical property is the nucleotidesequence of one or each self-complementary sequence.

In some embodiments, the physiochemical property is the extent ofmultimerization of one or more replication products. In someembodiments, the physiochemical property is the stoichiometry ofmonomeric and/or multimeric forms of the replication product in thenucleic acid preparation.

In some embodiments, the physiochemical property is the susceptibilityof one or more replication products to digestion with a restrictionendonuclease.

In some embodiments, the physiochemical property is the polarity ofmonomers in a dimeric form of the replication product, wherein thepolarity is head-to-head, head-to-tail or tail-to-tail.

In some embodiments, the physiochemical property is the molecular weightof one or more replication products or of a fragment of a replicationproduct. In some embodiments, the molecular weight is of a fragment ofthe one or more replication products that comprises one or eachself-complementary sequence. In some embodiments, the molecular weightis determined based on electrophoretic mobility. In some embodiments,the molecular weight is determined based on mass spectroscopy.

In some embodiments, the molecular weight is of a fragment of the one ormore replication products, and wherein prior to determining themolecular weight the fragment is amplified by a reaction comprisingprimer extension by a polymerase. In some embodiments, the reactioncomprising primer extension is a polymerase chain reaction.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows the theoretical secondary structure of AAV2 ITR based onmaximizing the stability and decreasing the Gibb's free energy (ΔG,negative values indicate spontaneous formation). FIG. 1B shows severalnon-limiting examples of truncations in the stem-region of the AAV2 ITRthat result in a nucleic acid molecule having asymmetric termini.

FIGS. 2A-2B show representations of symmetric and asymmetric open andclosed-ended duplex DNA (ceDNA) molecules. FIG. 2A shows severalnon-limiting examples of nucleic acids having symmetric termini (leftbox) and several non-limiting examples of nucleic acids havingasymmetric termini (e.g., ceDNA) (right box). FIG. 2B shows asymmetricterminal regions induce alteration of nucleic acid nicking and strandseparation induced during viral replication, resulting in formation ofclosed-ended duplex DNA molecules (right).

FIG. 3 shows a graphic depiction of a pair of asymmetric ITRs. A fulllength AAV2 ITR is shown on top, and an AAV2 ITR having a truncation inthe C-stem is depicted on the bottom. Both the full-length and thetruncated ITR comprise an operative Rep-binding element (RBE) and anoperative terminal resolution site (trs).

FIG. 4 shows non-limiting examples of nucleic acid constructs havingasymmetric self-complementary nucleic acid sequences (e.g., AAV2 ITRs)and a transgene associated with ocular disease.

FIG. 5 shows non-limiting examples of nucleic acid constructs havingasymmetric self-complementary nucleic acid sequences (e.g., AAV2 ITRs)and a transgene associated with blood disease.

FIG. 6 shows non-limiting examples of nucleic acid constructs havingasymmetric self-complementary nucleic acid sequences (e.g., AAV2 ITRs)and a transgene associated with liver disease.

FIG. 7 shows non-limiting examples of nucleic acid constructs havingasymmetric self-complementary nucleic acid sequences (e.g., AAV2 ITRs)and a transgene associated with lung disease.

FIGS. 8A-8D show delivery of ceDNA to the eye. Adult mice wereanesthetized by Ketamine/Xylazine (100/10 mg/kg) and the transfectionagent was delivered intravitreally by a trans scleral injection of avolume of 1-2 μl. Antibiotic ointment was applied on cornea to preventeye from drying while the mouse was recovering. Mouse was allowed torecover at 37 degree and then placed back into the mouse room for 2weeks and the euthanized by CO₂ asphyxiation. Retina was dissected andprocessed for flat mount or section. No GFP antibody staining wasnecessary to detect transfected cells. FIG. 8A shows a flat mount of GFPfluorescence on a mouse retina. FIG. 8B shows GFP fluorescence in across section of the retina. FIG. 8C shows GFP fluorescence and glialcell staining in a cross section of the retina. FIG. 8D shows GFPfluorescence in mouse retina after delivery of ceDNA (e.g., ceDNA havingasymmetric interrupted self-complementary sequences) by sub-retinalelectroporation (top) and intravitreal injection (bottom).

FIGS. 9A-9C show intracranial injection of ceDNA-GFP (e.g., ceDNA havingasymmetric interrupted self-complementary sequences and encoding GFP)formulated with in vivo jetPEI, into rat striatum. FIG. 9A showsstaining for GFP expression at 3 wks and 20 wks post-injection; similarGFP expression was seen at 3 wks and 20 wks. FIGS. 9B and 9C showimmunohistochemistry (IHC) with antibodies (Abs) against Iba1 (FIG. 9B)and MHCII (FIG. 9C); at 3 wks, no MHCII or Iba1 antigen was detected inthe brain sections.

FIGS. 10A-10B show results of a sequence analysis of the plasmid DNAinterrupted self-complementary sequences. FIG. 10C shows agarose gelelectrophoretic separation of ceDNA-GFP. The left side of the figureshows native gel electrophoresis of uncut ceDNA-GFP and ceDNA-GFPdigested with XhoI. Monomeric (˜2.1 kb) and dimeric (˜4.1 kb) conformerproducts are observed in the native gel; the 0.4 terminal fragment isobscured by fluorescence of impurities at the bottom of the gel. Theright side of the figure shows denaturing gel electrophoresis of uncutceDNA-GFP and ceDNA-GFP digested with XhoI. The dimeric (˜4.1 kb)conformer product is observed in the denaturing gel; denatured terminal0.4 kb products that run as a single stranded DNA at 0.8 kb are alsoobserved.

DETAILED DESCRIPTION

The disclosure relates in some aspects to compositions and methods fordelivery of a transgene to a subject (e.g., a cell of a subject, or atissue of a subject). The disclosure relates, in part, to the discoverythat replication of nucleic acids encoding a heterologous nucleic acidinsert flanked by certain types of asymmetric terminal sequences (e.g.,asymmetric interrupted self-complementary sequences) results in covalentlinkage of the asymmetric terminal sequences and leads to the productionof a novel conformation of closed-ended linear duplex DNA (ceDNA). Insome embodiments, nucleic acids having asymmetric interruptedself-complementary sequences may have improved expression, replication(e.g., production yield) in a subject compared to currently used genetherapy vectors. In some embodiments, improved expression of nucleicacids comprising asymmetric interrupted self-complementary sequences isrelated to a preference of RecQ helicases (e.g., RecQ1) to interact withnucleic acids comprising symmetric interrupted self-complementarysequences compared to nucleic acids having asymmetric interruptedself-complementary sequences.

In some embodiments, nucleic acids having asymmetric interruptedself-complementary sequences (e.g., are derived from a differentorganism or viral serotype, or are derived from the same organism orviral serotype but have different lengths, or a combination of theforegoing) may have reduced likelihood of insertional mutagenesis in asubject compared to currently used gene therapy vectors. In someembodiments, administration of nucleic acids having asymmetricinterrupted self-complementary sequences induce a reduced immuneresponse, or do not induce a detectable immune response, in a subjectrelative to plasmid DNA vectors.

A “nucleic acid” sequence refers to a DNA or RNA sequence. In someembodiments, proteins and nucleic acids of the disclosure are isolated.As used herein, the term “isolated” means artificially produced. In someembodiments, with respect to nucleic acids, the term “isolated” refersto a nucleic acid that is: (i) amplified in vitro by, for example,polymerase chain reaction (PCR); (ii) recombinantly produced bymolecular cloning; (iii) purified, as by restriction endonucleasecleavage and gel electrophoretic fractionation, or columnchromatography; or (iv) synthesized by, for example, chemical synthesis.An isolated nucleic acid is one which is readily manipulable byrecombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art. As used herein with respect to proteins or peptides, the term“isolated” refers to a protein or peptide that has been isolated fromits natural environment or artificially produced (e.g., by chemicalsynthesis, by recombinant DNA technology, etc.).

The skilled artisan will also realize that conservative amino acidsubstitutions may be made to provide functionally equivalent variants,or homologs of the capsid proteins. In some aspects the disclosureembraces sequence alterations that result in conservative amino acidsubstitutions. As used herein, a conservative amino acid substitutionrefers to an amino acid substitution that does not alter the relativecharge or size characteristics of the protein in which the amino acidsubstitution is made. Variants can be prepared according to methods foraltering polypeptide sequence known to one of ordinary skill in the artsuch as are found in references that compile such methods, e.g.,Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, etal., eds., John Wiley & Sons, Inc., New York. For example, in someembodiments, conservative substitutions of amino acids includesubstitutions made among amino acids within the following groups: (a) M,I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g)E, D. Therefore, one can make conservative amino acid substitutions tothe amino acid sequence of proteins and polypeptides disclosed herein.

Interrupted Self-Complementary Sequences

The disclosure is based, in part, on the discovery that nucleic acidshaving asymmetric terminal sequences (e.g., asymmetric interruptedself-complementary sequences) form closed-ended linear duplex DNAstructures (e.g., ceDNA) that, in some embodiments, exhibit reducedimmunogenicity compared to currently available gene delivery vectors. Insome embodiments, ceDNA behaves as linear duplex DNA under nativeconditions and transforms into single-stranded circular DNA underdenaturing conditions. Without wishing to be bound by any particulartheory, nucleic acids described by the disclosure (e.g., ceDNA) areuseful, in some embodiments, for the delivery of heterologous nucleicacid inserts (e.g., transgenes) to a subject.

In some aspects, the disclosure provides a nucleic acid comprising aheterologous nucleic acid insert flanked by interruptedself-complementary sequences, each self-complementary sequence having anoperative terminal resolution site and a rolling circle replicationprotein binding element, wherein one self-complementary sequence isinterrupted by a cross-arm sequence forming two opposing,lengthwise-symmetric stem-loops, each of the opposinglengthwise-symmetric stem-loops having a stem portion and a loopportion, wherein the other of the self-complementary sequences isinterrupted by a truncated cross-arm sequence.

As used herein, the term “flanked” refers to the positioning of a firstinterrupted self-complementary sequence upstream (e.g., 5′) relative toa heterologous nucleic acid insert and a second interruptedself-complementary sequence downstream (e.g., 3′) relative to theheterologous nucleic acid insert. For example, an adeno-associated virusgenome comprises open reading frames of the rep and cap genes “flanked”by inverted terminal repeats (ITRs).

As used herein, the term “interrupted self-complementary sequence”refers to a polynucleotide sequence encoding a nucleic acid havingpalindromic (e.g., a contiguous stretch of polynucleotides that isidentical to its complementary strand, if both are “read” in the same 5′to 3′ direction) terminal sequences that are interrupted by one or morestretches of non-palindromic polynucleotides. Generally, apolynucleotide encoding one or more interrupted palindromic sequenceswill fold back upon itself, forming a stem-loop structure (e.g., ahairpin loop, a “T”-shaped loop, or a “Y”-shaped loop), for example asshown in the AAV2 ITR structure depicted in FIG. 1A and the exemplarystructures depicted in FIG. 2A.

In some embodiments, an interrupted self-complementary sequence forms a“T”-shaped structure having a stem sequence and a cross-arm sequence. Insome embodiments, the “cross-arm sequence” forms two opposing (e.g.,relative to the stem sequence), lengthwise-symmetric stem-loops, each ofthe opposing lengthwise-symmetric stem-loops having a stem portion and aloop portion. For example, in some embodiments, the stem sequence isformed by hybridization of the complementary (e.g., palindromic) 5′- and3′-ends of a polynucleotide sequence (referred to as “A-A′”), where theA-A′ palindrome is interrupted by the cross-arm polynucleotide sequenceformed by a pair of loop-forming interrupted palindromic sequencesdenoted “B-B′” and “C-C′”, respectively, as shown in FIG. 1A. In someembodiments, the loop portion of each cross arm (e.g., loops formed byinterrupted palindromic sequences B-B′ and C-C′) is formed from unpairednucleotides (e.g., unpaired deoxyribonucleotides). It should beappreciated that an interrupted self-complementary sequence described bythe disclosure may comprise more than two (e.g., 3, 4, or more)cross-arm sequences.

An interrupted self-complementary sequence can be of any size, providedthat the sequence forms a hairpin loop and functions as a primer fornucleic acid replication (e.g., DNA replication). For example, aninterrupted self-complementary sequence can range from about 20 to about2000 nucleotides in length. In some embodiments, an interruptedself-complementary sequence ranges from about 40 to 1000 nucleotides inlength. In some embodiments, an interrupted self-complementary sequenceis at least 40, at least 50, at least 60, at least 70, at least 80, atleast 90, at least 100, at least 200, at least 300, at least 400, atleast 500, at least 600, at least 700, at least 800, at least 900, or upto 1000 nucleotides in length. In some embodiments, an interruptedself-complementary sequence is more than 1000 nucleotides in length. Insome embodiments, an interrupted self-complementary sequence ranges fromabout 100 to 160 nucleotides in length. In some embodiments, aninterrupted self-complementary nucleotide ranges from about 115 to about150 nucleotides in length.

In some aspects, the disclosure relates to nucleic acid having aninterrupted self-complementary sequence that forms opposinglengthwise-symmetric stem-loops. In some embodiments, each of theopposing lengthwise-symmetric stem-loops have a stem portion in therange of 3 to 15 base pairs in length. In some embodiments, each of theopposing lengthwise-symmetric stem-loops have a stem portion in therange of 8 to 10 base pairs in length. In some embodiments, each of theopposing lengthwise-symmetric stem-loops have a stem portion that is 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

Generally, a loop portion of a stem-loop structure comprises at least 2unpaired nucleotides. In some embodiments, each loop portion has 2 to 5unpaired deoxyribonucleotides (e.g., 2, 3, 4, or 5 unpaireddeoxyribonucleotides). In some embodiments, each loop portion of across-arm sequence described by the disclosure has threedeoxyribonucleotides. In some embodiments, one loop portion of across-arm sequence described by the disclosure has three deoxythymidinesand the other loop portion has three deoxyadenosines.

In some aspects, the disclosure relates to interruptedself-complementary sequences flanking a nucleic acid insert, where theinterrupted self-complementary sequences are asymmetric with respect toone another (e.g., are derived from a different organism or viralserotype, or are derived from the same organism or viral serotype buthave different lengths, or a combination of the foregoing). In someembodiments, one of a pair of asymmetric self-complementary sequencescomprises a truncated cross-arm sequence. As used herein, “truncatedcross-arm sequence” refers to a cross-arm sequence that has a shorterlength relative to the corresponding self-complementary sequenceflanking a heterologous nucleic acid sequence. A truncated cross-armsequence can have between 1 and 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or 50) nucleotide deletions relative to a full-lengthcross-arm sequence. In some embodiments, a truncated cross-arm sequencehas between 1 and 30 nucleotide deletions relative to a full-lengthcross-arm sequence. In some embodiments, a truncated cross-arm sequencecontains between 2 and 20 nucleotide deletions relative to a full-lengthcross-arm sequence.

In some embodiments, a truncated cross-arm sequence forms two opposing,lengthwise-asymmetric stem-loops. In some embodiments, one of theopposing, lengthwise-asymmetric stem-loops of the truncated cross-armsequence has a stem portion in the range of 8 to 10 base pairs in lengthand a loop portion having 2 to 5 unpaired deoxyribonucleotides. In someembodiments, a one lengthwise-asymmetric stem-loop of a truncatedcross-arm sequence has a stem portion less than 8 base pairs in lengthand a loop portion having 2 to 5 deoxyribonucleotides. In someembodiments, the one lengthwise-asymmetric stem-loop has a stem portionless than 3 base pairs in length. In some embodiments, the onelengthwise-asymmetric stem-loops has a loop portion having 3 or fewerdeoxyribonucleotides.

Generally, a truncated cross-arm sequence does not contain anynucleotide deletions (e.g., relative to a non-truncated cross-armsequence) in the A or A′ regions, so as not to interfere with DNAreplication (e.g., binding to a RBE by Rep protein, or nicking at aterminal resolution site). In some embodiments, a truncated cross-armsequence has one or more deletions in the B, B′, C, and/or C′ region.Several non-limiting examples of truncated cross-arm sequences are shownbelow:

AAV2 ITR Δ C-region indicated by parenthesis; all or partial deletionswithin the square brackets can be used to create asymmetric interruptedself-complementary sequences; below, “RBE′” refers to “Rep-bindingelement”.       C-Region                    B-Region              Δ                          RBE′5′cggg(cgaccaaaggtc)gcccg-a-cgcccgggctttgcccgggc (SEQ ID NO: 2)5′cggg(cgaccaaaggtcg)cccg-a-cgcccgggctttgcccgggc (SEQ ID NO: 2)5′gccc(gggcaaagccc)gggcg-t-cgggcgacctttggtcgcccg (SEQ ID NO: 3)5′gccc(gggcaaagccc)gggcg-t-cgggcgacctttggtcgcccg (SEQ ID NO: 3)5′[cgggcgaccaaaggtcgcccg]-a-cgcccgggctttgcccgggc (SEQ ID NO: 2)5′[cgggcgaccaaaggtcgcccg]-a-cgcccgggctttgcccgggc (SEQ ID NO: 2)5′[gcccgggcaaagcccgggcg]-t-cgggcgacctttggtcgcccg (SEQ ID NO: 3)5′[gcccgggcaaagcccgggcg]-t-cgggcgacctttggtcgcccg (SEQ ID NO: 3)Generally, the thermodynamic properties of a nucleic acid (e.g., Gibbsfree energy (ΔG), G+C composition, A+T composition, melting temperature,base composition of each strand, length of complementary sequence,unpaired bases within the duplex region, and unpaired bases constitutingthe loop) required for hairpin formation are known in the art, forexample as disclosed in Bosco et al., Nucl. Acids Res. (2013) doi:10.1093/nar/gkt1089; First published online: Nov. 12, 2013.

In some embodiments, the cross-arm sequence has a Gibbs free energy (ΔG)of unfolding under physiological conditions in the range of −12 kcal/molto −30 kcal/mol. In some embodiments, the cross-arm sequence has a Gibbsfree energy (ΔG) of unfolding under physiological conditions in therange of −20 kcal/mol to −25 kcal/mol. In some embodiments, thethermodynamic properties of a truncated cross-arm sequence can be thesame (e.g., identical) relative to a full-length cross-arm sequence eventhough they may have sequence differences that render them asymmetric.In some embodiments, the thermodynamic properties of a truncatedcross-arm sequence can be the different than those of a full-lengthcross-arm sequence. For example, in some embodiments, a truncatedcross-arm sequence has a Gibbs free energy (ΔG) of unfolding underphysiological conditions in the range of 0 kcal/mol to greater than −22kcal/mol.

As used herein, the term “operative” refers to the ability of a nucleicacid sequence to perform its intended function. For example, an“operative binding region” is a nucleic acid sequence that retainsbinding function for its intended target (e.g., a protein or nucleicacid). In another example, an “operative cleavage site” is a nucleicacid sequence that retains its ability to be specifically cleaved by aparticular enzyme or enzymes.

Aspects of the disclosure relate to the discovery thatself-complementary nucleic acid sequences comprising an operativerolling circle replication protein binding element is required for theformation of closed-ended linear duplex DNA (ceDNA). As used here,“rolling circle replication protein binding element” refers to aconserved nucleic acid sequence (e.g., motif) that is recognized andbound by a rolling circle replication protein, which is a viralnonstructural protein (NS protein) that initiates rolling circle (e.g.,rolling hairpin) replication. Rolling circle (e.g., rolling hairpin)replication is described by Tattersall et al. Nature 2009, 263, pp.106-109. Examples of NS proteins include, but are not limited to AAV Repproteins (e.g., Rep78, Rep68, Rep52, Rep40), parvovirus nonstructuralproteins (e.g., NS2), rotavirus nonstructural proteins (e.g., NSP1), anddensovirus nonstructural proteins (e.g., PfDNV NS1). In someembodiments, the rolling circle replication protein binding element is aRep binding element (RBE). In some embodiments, the RBE comprises thesequence 5′-GCTCGCTCGCTC-3′.

In some embodiments, rolling circle replication proteins are from thedependoparvovirus genus of the Parvoviridae family of viruses withlinear single-stranded DNA genomes. In some embodiments, the rollingcircle replication proteins are from the genera of the autonomousParvovirinae including mice minute virus, Aleutian mink disease virus,bovine parvovirus, canine parvovirus, chicken parvovirus, felinepanleukopenia virus, feline parvovirus, goose parvovirus, HB parvovirus,H-1 parvovirus, Kilham rat virus, lapine parvovivirus, LUIII virus, minkenteritis virus, mouse parvovirus, porcine parvovirus, raccoonparvovivurs, RT parvovirus, Tumor virus X, rat parvovirus 1a, barbarieduck parvovirus, equine parvovirus, hamster parvovirus, and rheumatoridarthritis virus 1. In some embodiments, the genus is parvovirus. In someembodiments, the rolling circle replication proteins are from the generaof Densovirinae including brevidensovirus, densovirus, and iteravirus.

In some embodiments, a rolling circle replication protein is from thegenera of the subfamily Parvovirinae. Examples of Parvovirinae generainclude but are not limited to Amdoparvovirus, Aveparvovirus,Bocaparvovirus, Copiparvovirus, Dependoparvovirus, Erythroparvovirus,Protoparvovirus, Tetraparvovirus. In some embodiments, the rollingcircle replication proteins are from the genera of the subfamily,Densovirinae. Examples of Densovirinae genera include but are notlimited to Amdoparvovirus, Aveparvovirus, Bocaparvovirus,Copiparvovirus, Dependoparvovirus, Erythroparvovirus, Protoparvovirus,and Tetraparvovirus. In some embodiments, the rolling circle replicationprotein(s) is from a Dependovirus, such as Adeno-associated virus 2(AAV2), Adeno-associated virus 3 (AAV3), Adeno-associated virus 4(AAV4), or Adeno-associated virus 5 (AAV5), or any combination thereof.

In some embodiments, the rolling circle replication proteins are derivedfrom the single-stranded DNA bacteriophage families. In someembodiments, the virus families are the Microviridae and the Inoviridae.

In some embodiments, the rolling circle replication proteins are derivedfrom Gram positive bacteria.

Aspects of the disclosure relate to the discovery that interruptedself-complementary nucleic acid sequences comprising an operativeterminal resolution site (trs) are required for the formation ofclosed-ended linear duplex DNA (ceDNA). Typically, replication ofnucleic acids comprising interrupted self-complementary nucleic acidsequences (e.g., AAV ITRs) is initiated from the 3′ end of the cross-arm(e.g., hairpin structure) and generates a duplex molecule in which oneof the ends is covalently closed; the covalently closed ends of theduplex molecule are then cleaved by a process called terminal resolutionto form a two separate single-stranded nucleic acid molecules. Withoutwishing to be bound by any particular theory, the process of terminalresolution is mediated by a site- and strand-specific endonucleasecleavage at a terminal resolution site (trs) (e.g., a rolling circlereplication protein, such as AAV Rep protein). Examples of trs sequencesinclude 3′-CCGGTTG-5 and 5′-AGTTGG-3′ (recognized by AAV2 p5 protein).It has been hypothesized that Rep-mediated strand nicking takes placebetween the central di-thymidine (“TT”) portion of the trs sequence.Therefore, in some embodiments, the operative terminal resolution sitecomprises a sequence 5′-TT-3′.

Aspects of the disclosure relate to the positioning of a terminalresolution site (trs) relative to a rolling circle replication proteinbinding element. Generally, a trs is positioned upstream (e.g., 5′)relative to a rolling circle replication protein binding element.However, in some embodiments, a trs is positioned downstream (e.g., 3′)relative to a rolling circle replication protein binding element. Insome embodiments, the 3′ end of the operative terminal resolution siteis 15 to 25 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 nucleotides) from the 5′ end of the rolling circle replicationprotein binding element.

In some embodiments, an interrupted self-complementary sequence is anAAV inverted terminal repeat sequence. The AAV ITR sequence can be ofany AAV serotype, including but not limited to, AAV1, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, non-human primate AAV serotypes (e.g.,AAVrh.10), and variants thereof. In some embodiments, an interruptedself-complementary sequence is an AAV2 ITR or a variant thereof (e.g.,AAV2 ITR, or a truncated AAV2 ITR having a deletion in the “B arm” or “Carm”). In some embodiments, an interrupted self-complementary sequenceis an AAV5 ITR or a variant thereof (e.g., AAV5 ITR, or a truncated AAV5ITR having a deletion in the “B arm” or “C arm”). As used herein, a“variant” of an AAV ITR is a polynucleotide having between about 70% andabout 99.9% similarity to a wild-type AAV ITR sequence. In someembodiments, an AAV ITR variant is about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, or about 99% identical to a wild-typeAAV ITR.

An AAV ITR can exist in two conformations: “flip” and “flop”, which area result of the rolling hairpin mechanism of AAV replication. Anon-limiting example of an interrupted self-complementary sequence(e.g., AAV2 ITR) in both “flip” and “flop” conformations is shown below:

GenBank (>gi|110645916|ref|NC_001401.2|Adeno-associated virus - 2, complete genome) Flop conformation(SEQ ID NO: 4) ttggccactccctctctgcgcgctcgctcgctcactgaggc_cgggcgaccaaaggtcgcccg acgcccgggctttg||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||| | | aaccggtgagggagagacgcgcgagcgagcgagtgactccg_ gcccgctggtttccagcgggctgcgggcccgaaac (SEQ ID NO: 5) cccgggc_ggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||ggg cccg_ccggagtcactcgctcgctcgcgcgtctctccctcaccggttgaggtagtgatccccaaggaFlip conformation (SEQ ID NO: 6)aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggcc_gggcgaccaaaggt||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||tccttggggatcactacctcaaccggtgagggagagacgcgcgagcgagcgagtgactccgg_cccgctggtttcca(SEQ ID NO: 7)cgcccgacgcccgggctttgcccgggcg_gcctcagtgagcgagcgagcgcgcagagagggagtggccaa|||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||gcgggctgcgggcccgaaacgggcccgc_cggagtcactcgctcgctcgcgcgtctctccctcaccggttIn some embodiments, a nucleic acid described by the disclosure (e.g.,ceDNA) comprises an interrupted self-complementary sequence in the flipconformation. In some embodiments, a nucleic acid described by thedisclosure (e.g., ceDNA) comprises an interrupted self-complementarysequence in the flop conformation.

Aspects of the disclosure relate to compositions comprising a populationof nucleic acids described by the disclosure. Generally, the populationsmay be homogenous (e.g., comprising multiple copies of the same nucleicacid) or heterogeneous (e.g., comprising multiple different nucleicacids). For example, in some embodiments, a composition comprises amonomeric nucleic acid (e.g., a population of a single species ofmonomeric nucleic acid) comprising a single subunit. In someembodiments, the subunit comprises a heterologous nucleic acid insertflanked by interrupted self-complementary sequences, eachself-complementary sequence having an operative terminal resolution siteand a rolling circle replication protein binding element, wherein oneself-complementary sequence is interrupted by a cross-arm sequenceforming two opposing, lengthwise-symmetric stem-loops and the other ofthe self-complementary sequences is interrupted by a truncated cross-armsequence.

In some embodiments, a composition comprises a multimeric nucleic acidcomprising two or more subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore subunits). In some embodiments, each subunit of the multimericnucleic acid comprises a heterologous nucleic acid insert flanked byinterrupted self-complementary sequences, each self-complementarysequence having an operative terminal resolution site and a rollingcircle replication protein binding element, wherein oneself-complementary sequence is interrupted by a cross-arm sequenceforming two opposing, lengthwise-symmetric stem-loops and the other ofthe self-complementary sequences is interrupted by a truncated cross-armsequence. In some embodiments, the multimeric nucleic acid comprises twosubunits (e.g., is a dimer). In some embodiments, each multimer has atleast one, and in some cases only one, self-complementary terminalpalindrome.

In some embodiments, the subunits of a multimeric nucleic acid formconcatamers. As used herein, “concatamer” refers to a nucleic acidmolecule comprising multiple copies of the same or substantially thesame nucleic acid sequences (e.g., subunits) that are typically linkedin a series. In some embodiments, concatamers described by thedisclosure can be orientated in either a head-to-head polarity, or atail-to-tail polarity. In embodiments in which subunits contain aheterologous nucleic acid sequence configured to express an RNAtranscript, “head-to-head” polarity refers to a concatamer in which theinterrupted self-complementary sequences closest to the promotersequence of each subunit are covalently linked (e.g., the subunits arelinked 5′-end to 5′-end). In such embodiments, “tail-to-tail” polarityrefers to a concatamer in which the interrupted self-complementarysequences distal to the promoter sequence of each subunit are covalentlylinked (e.g., the subunits are linked 5′-end to 5′-end). In someembodiments, the two subunits are linked in a tail-to-tail configuration(e.g., polarity).

In some embodiments, a composition comprises both monomeric andmultimeric nucleic acids (e.g., comprises a heterogeneous population ofnucleic acids).

Heterologous Nucleic Acid Inserts

The composition of the transgene sequence (e.g., heterologous nucleicacid insert) of the nucleic acid will depend upon the use to which theresulting nucleic acid will be put. For example, one type of transgenesequence includes a reporter sequence, which upon expression produces adetectable signal. In another example, the transgene encodes atherapeutic protein or therapeutic functional RNA. In another example,the transgene encodes a protein or functional RNA that is intended to beused for research purposes, e.g., to create a somatic transgenic animalmodel harboring the transgene, e.g., to study the function of thetransgene product. In another example, the transgene encodes a proteinor functional RNA that is intended to be used to create an animal modelof disease. Appropriate transgene coding sequences will be apparent tothe skilled artisan.

The disclosure is based, in part, on the discovery that unlike AAVvectors, nucleic acids described herein (e.g., ceDNA) are not limitedwith respect to the size of a heterologous nucleic acid insert (e.g.,transgene sequence). In some embodiments, a transgene (e.g.,heterologous nucleic acid insert) flanked by interruptedself-complementary sequences ranges from about 10 to about 5,000 basepairs, about 10 to about 10,000 base pairs, about 10 to about 50,000base pairs in length. In some embodiments, a transgene (e.g.,heterologous nucleic acid insert) flanked by interruptedself-complementary sequences ranges from about 10 to about 50 base pairsin length. In some embodiments, a transgene (e.g., heterologous nucleicacid insert) flanked by interrupted self-complementary sequences rangesfrom about 20 to about 100 base pairs in length. In some embodiments, atransgene (e.g., heterologous nucleic acid insert) flanked byinterrupted self-complementary sequences ranges from about 500 to about1500 base pairs in length. In some embodiments, a transgene (e.g.,heterologous nucleic acid insert) flanked by interruptedself-complementary sequences ranges from about 1000 to about 5000 basepairs in length. In some embodiments, the size of a transgene (e.g.,heterologous nucleic acid insert) exceeds the capacity of a traditionalAAV vector (e.g., exceeds about 4.8 kb).

Reporter sequences that may be provided in a transgene include, withoutlimitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ),alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), luciferase, and others wellknown in the art. When associated with regulatory elements which drivetheir expression, the reporter sequences, provide signals detectable byconventional means, including enzymatic, radiographic, colorimetric,fluorescence or other spectrographic assays, fluorescent activating cellsorting assays and immunological assays, including enzyme linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) andimmunohistochemistry. For example, where the marker sequence is the LacZgene, the presence of the vector carrying the signal is detected byassays for β-galactosidase activity. Where the transgene is greenfluorescent protein or luciferase, the vector carrying the signal may bemeasured visually by color or light production in a luminometer. Suchreporters can, for example, be useful in verifying the tissue-specifictargeting capabilities and tissue specific promoter regulatory activityof a nucleic acid.

In some aspects, the disclosure provides nucleic acids for use inmethods of preventing or treating one or more genetic deficiencies ordysfunctions in a mammal, such as for example, a polypeptide deficiencyor polypeptide excess in a mammal, and particularly for treating orreducing the severity or extent of deficiency in a human manifesting oneor more of the disorders linked to a deficiency in such polypeptides incells and tissues. The method involves administration of nucleic acid(e.g., a nucleic acid as described by the disclosure) that encodes oneor more therapeutic peptides, polypeptides, siRNAs, microRNAs, antisensenucleotides, etc. in a pharmaceutically-acceptable carrier to thesubject in an amount and for a period of time sufficient to treat thedeficiency or disorder in the subject suffering from such a disorder.

Thus, the disclosure embraces the delivery of nucleic acids (e.g.,nucleic acids as described by the disclosure) encoding one or morepeptides, polypeptides, or proteins, which are useful for the treatmentor prevention of disease states in a mammalian subject. Exemplarytherapeutic proteins include one or more polypeptides selected from thegroup consisting of growth factors, interleukins, interferons,anti-apoptosis factors, cytokines, anti-diabetic factors, anti-apoptosisagents, coagulation factors, anti-tumor factors. Other non-limitingexamples of therapeutic proteins include BDNF, CNTF, CSF, EGF, FGF,G-SCF, GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF,VEGF, TGF-B2, TNF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(187A), viral IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16 IL-17, and IL-18.

The nucleic acids (e.g., nucleic acids as described by the disclosure)may comprise a gene to be transferred (e.g., expressed in) to a subjectto treat a disease associated with reduced expression, lack ofexpression or dysfunction of the gene. Exemplary genes and associateddisease states include, but are not limited to: glucose-6-phosphatase,associated with glycogen storage deficiency type 1A;phosphoenolpyruvate-carboxykinase, associated with Pepck deficiency;galactose-1 phosphate uridyl transferase, associated with galactosemia;phenylalanine hydroxylase, associated with phenylketonuria; branchedchain alpha-ketoacid dehydrogenase, associated with Maple syrup urinedisease; fumarylacetoacetate hydrolase, associated with tyrosinemia type1; methylmalonyl-CoA mutase, associated with methylmalonic acidemia;medium chain acyl CoA dehydrogenase, associated with medium chain acetylCoA deficiency; omithine transcarbamylase, associated with omithinetranscarbamylase deficiency; argininosuccinic acid synthetase,associated with citrullinemia; low density lipoprotein receptor protein,associated with familial hypercholesterolemia;UDP-glucouronosyltransferase, associated with Crigler-Najjar disease;adenosine deaminase, associated with severe combined immunodeficiencydisease; hypoxanthine guanine phosphoribosyl transferase, associatedwith Gout and Lesch-Nyan syndrome; biotinidase, associated withbiotinidase deficiency; beta-glucocerebrosidase, associated with Gaucherdisease; beta-glucuronidase, associated with Sly syndrome; peroxisomemembrane protein 70 kDa, associated with Zellweger syndrome;porphobilinogen deaminase, associated with acute intermittent porphyria;alpha-1 antitrypsin for treatment of alpha-1 antitrypsin deficiency(emphysema); erythropoietin for treatment of anemia due to thalassemiaor to renal failure; vascular endothelial growth factor, angiopoietin-1,and fibroblast growth factor for the treatment of ischemic diseases;thrombomodulin and tissue factor pathway inhibitor for the treatment ofoccluded blood vessels as seen in, for example, atherosclerosis,thrombosis, or embolisms; aromatic amino acid decarboxylase (AADC), andtyrosine hydroxylase (TH) for the treatment of Parkinson's disease; thebeta adrenergic receptor, anti-sense to, or a mutant form of,phospholamban, the sarco(endo)plasmic reticulum adenosinetriphosphatase-2 (SERCA2), and the cardiac adenylyl cyclase for thetreatment of congestive heart failure; a tumor suppessor gene such asp53 for the treatment of various cancers; a cytokine such as one of thevarious interleukins for the treatment of inflammatory and immunedisorders and cancers; dystrophin or minidystrophin and utrophin orminiutrophin for the treatment of muscular dystrophies; and, insulin forthe treatment of diabetes.

In some embodiments, the disclosure relates to a heterologous nucleicacid insert encoding a protein or functional RNA useful for thetreatment of a condition, disease or disorder associated with thecentral nervous system (CNS). The following is a non-limiting list ofgenes associated with CNS disease: DRD2, GRIA1, GRIA2, GRIN1, SLC1A1,SYP, SYT1, CHRNA7, 3Rtau/4rTUS, APP, BAX, BCL-2, GRIK1, GFAP, IL-1,AGER, associated with Alzheimer's Disease; UCH-L1, SKP1, EGLN1, Nurr-1,BDNF, TrkB, gstm1, S106β, associated with Parkinson's Disease; IT15,PRNP, JPH3, TBP, ATXN1, ATXN2, ATXN3, Atrophin 1, FTL, TITF-1,associated with Huntington's Disease; FXN, associated with Freidrich'sataxia; ASPA, associated with Canavan's Disease; DMD, associated withmuscular dystrophy; and SMN1, UBE1, DYNC1H1 associated with spinalmuscular atrophy. In some embodiments, the disclosure relates to aheterologous nucleic acid insert that expresses one or more of theforegoing genes or fragments thereof. In some embodiments, thedisclosure relates to a heterologous nucleic acid insert that expressesone or more functional RNAs that inhibit expression of one or more ofthe foregoing genes.

In some embodiments, the disclosure relates to a heterologous nucleicacid insert encoding a protein or functional RNA useful for thetreatment of a condition, disease or disorder associated with thecardiovascular system. The following is a non-limiting list of genesassociated with cardiovascular disease: VEGF, FGF, SDF-1, connexin 40,connexin 43, SCN4a, HIF1α, SERCa2a, ADCY1, and ADCY6. In someembodiments, the disclosure relates to a heterologous nucleic acidinsert that expresses one or more of the foregoing genes or fragmentsthereof. In some embodiments, the disclosure relates to a heterologousnucleic acid insert that expresses one or more functional RNAs thatinhibit expression of one or more of the foregoing genes.

In some embodiments, the disclosure relates to a heterologous nucleicacid insert encoding a protein or functional RNA useful for thetreatment of a condition, disease or disorder associated with thepulmonary system. The following is a non-limiting list of genesassociated with pulmonary disease: CFTR, AAT, TNFα, TGFβ1, SFTPA1,SFTPA2, SFTPB, SFTPC, HPS1, HPS3, HPS4, ADTB3A, IL1A, IL1B, LTA, IL6,CXCR1, and CXCR2. In some embodiments, the disclosure relates to aheterologous nucleic acid insert that expresses one or more of theforegoing genes or fragments thereof. In some embodiments, thedisclosure relates to a heterologous nucleic acid insert that expressesone or more functional RNAs that inhibit expression of one or more ofthe foregoing genes. Non-limiting examples of heterologous nucleic acidinserts encoding a protein or functional RNA useful for the treatment ofa condition, disease or disorder associated with the pulmonary systemare depicted in FIG. 10.

In some embodiments, the disclosure relates to a heterologous nucleicacid insert encoding a protein or functional RNA useful for thetreatment of a condition, disease or disorder associated with the liver.The following is a non-limiting list of genes associated with liverdisease: α1-AT, HFE, ATP7B, fumarylacetoacetate hydrolase (FAH),glucose-6-phosphatase, NCAN, GCKR, LYPLAL1, PNPLA3, lecithin cholesterolacetyltransferase, phenylalanine hydroxylase, and G6PC. In someembodiments, the disclosure relates to a heterologous nucleic acidinsert that expresses one or more of the foregoing genes or fragmentsthereof. In some embodiments, the disclosure relates to a heterologousnucleic acid insert that expresses one or more functional RNAs thatinhibit expression of one or more of the foregoing genes. Non-limitingexamples of heterologous nucleic acid inserts encoding a protein orfunctional RNA useful for the treatment of a condition, disease ordisorder associated with the liver are depicted in FIG. 9.

In some embodiments, the disclosure relates to a heterologous nucleicacid insert encoding a protein or functional RNA useful for thetreatment of a condition, disease or disorder associated with thekidney. The following is a non-limiting list of genes associated withkidney disease: PKD1, PKD2, PKHD1, NPHS1, NPHS2, PLCE1, CD2AP, LAMB2,TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, SCARB3, FN1, COL4A5, COL4A6,COL4A3, COL4A4, FOX1C, RET, UPK3A, BMP4, SIX2, CDC5L, USF2, ROBO2,SLIT2, EYA1, MYOG, SIX1, SIX5, FRAS1, FREM2, GATA3, KAL1, PAX2, TCF2,and SALL1. In some embodiments, the disclosure relates to a heterologousnucleic acid insert that expresses one or more of the foregoing genes orfragments thereof. In some embodiments, the disclosure relates to aheterologous nucleic acid insert that expresses one or more functionalRNAs that inhibit expression of one or more of the foregoing genes.

In some embodiments, the disclosure relates to a heterologous nucleicacid insert encoding a protein or functional RNA useful for thetreatment of a condition, disease or disorder associated with the eye.The following is a non-limiting list of genes associated with oculardisease: ABCA4, VEGF, CEP290, CFH, C3, MT-ND2, ARMS2, TIMP3, CAMK4,FMN1, RHO, USH2A, RPGR, RP2, TMCO, SIX1, SIX6, LRP12, ZFPM2, TBK1, GALC,myocilin, CYP1B1, CAV1, CAV2, optineurin and CDKN2B. In someembodiments, the disclosure relates to a heterologous nucleic acidinsert that expresses one or more of the foregoing genes or fragmentsthereof. In some embodiments, the disclosure relates to a heterologousnucleic acid insert that expresses one or more functional RNAs thatinhibit expression of one or more of the foregoing genes. Non-limitingexamples of heterologous nucleic acid inserts encoding a protein orfunctional RNA useful for the treatment of a condition, disease ordisorder associated with the eye are depicted in FIG. 7.

In some embodiments, the disclosure relates to a heterologous nucleicacid insert encoding a protein or functional RNA useful for thetreatment of a condition, disease or disorder associated with the blood(e.g., red blood cells). The following is a non-limiting list of genesassociated with diseases and disorders of the blood: Factor VIII(FVIII), Factor IX (FIX), von Willebrand factor (VWF). In someembodiments, the disclosure relates to a heterologous nucleic acidinsert that expresses one or more of the foregoing genes or fragmentsthereof. In some embodiments, the disclosure relates to a heterologousnucleic acid insert that expresses one or more functional RNAs thatinhibit expression of one or more of the foregoing genes. Non-limitingexamples of heterologous nucleic acid inserts encoding a protein orfunctional RNA useful for the treatment of a condition, disease ordisorder associated with the blood are depicted in FIG. 8.

The nucleic acids of the disclosure (e.g., nucleic acid having aheterologous nucleic acid insert) can be used to restore the expressionof genes that are reduced in expression, silenced, or otherwisedysfunctional in a subject (e.g., a tumor suppressor that has beensilenced in a subject having cancer). The nucleic acids of thedisclosure can also be used to knockdown the expression of genes thatare aberrantly expressed in a subject (e.g., an oncogene that isexpressed in a subject having cancer). In some embodiments, aheterologous nucleic acid insert encoding a gene product associated withcancer (e.g., tumor suppressors) may be used to treat the cancer, byadministering nucleic acid comprising the heterologous nucleic acidinsert to a subject having the cancer. In some embodiments, a nucleicacid comprising a heterologous nucleic acid insert encoding a smallinterfering nucleic acid (e.g., shRNAs, miRNAs) that inhibits theexpression of a gene product associated with cancer (e.g., oncogenes)may be used to treat the cancer, by administering nucleic acidcomprising the heterologous nucleic acid insert to a subject having thecancer. In some embodiments, nucleic comprising a heterologous nucleicacid insert encoding a gene product associated with cancer (or afunctional RNA that inhibits the expression of a gene associated withcancer) may be used for research purposes, e.g., to study the cancer orto identify therapeutics that treat the cancer. The following is anon-limiting list of exemplary genes known to be associated with thedevelopment of cancer (e.g., oncogenes and tumor suppressors): AARS,ABCB1, ABCC4, ABI2, ABL1, ABL2, ACK1, ACP2, ACY1, ADSL, AK1, AKR1C2,AKT1, ALB, ANPEP, ANXA5, ANXA7, AP2M1, APC, ARHGAP5, ARHGEF5, ARID4A,ASNS, ATF4, ATM, ATP5B, ATP5O, AXL, BARD1, BAX, BCL2, BHLHB2, BLMH,BRAF, BRCA1, BRCA2, BTK, CANX, CAP1, CAPN1, CAPNS1, CAV1, CBFB, CBLB,CCL2, CCND1, CCND2, CCND3, CCNE1, CCT5, CCYR61, CD24, CD44, CD59, CDC20,CDC25, CDC25A, CDC25B, CDC2L5, CDK10, CDK4, CDK5, CDK9, CDKL1, CDKN1A,CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2D, CEBPG, CENPC1, CGRRF1, CHAF1A,CIB1, CKMT1, CLK1, CLK2, CLK3, CLNS1A, CLTC, COL1A1, COL6A3, COX6C,COX7A2, CRAT, CRHR1, CSF1R, CSK, CSNK1G2, CTNNA1, CTNNB1, CTPS, CTSC,CTSD, CUL1, CYR61, DCC, DCN, DDX10, DEK, DHCR7, DHRS2, DHX8, DLG3, DVL1,DVL3, E2F1, E2F3, E2F5, EGFR, EGR1, EIF5, EPHA2, ERBB2, ERBB3, ERBB4,ERCC3, ETV1, ETV3, ETV6, F2R, FASTK, FBN1, FBN2, FES, FGFR1, FGR, FKBP8,FN1, FOS, FOSL1, FOSL2, FOXG1A, FOXO1A, FRAP1, FRZB, FTL, FZD2, FZD5,FZD9, G22P1, GAS6, GCN5L2, GDF15, GNA13, GNAS, GNB2, GNB2L1, GPR39,GRB2, GSK3A, GSPT1, GTF2I, HDAC1, HDGF, HMMR, HPRT1, HRB, HSPA4, HSPA5,HSPA8, HSPB1, HSPH1, HYAL1, HYOU1, ICAM1, ID1, ID2, IDUA, IER3, IFITM1,IGF1R, IGF2R, IGFBP3, IGFBP4, IGFBP5, IL1B, ILK, ING1, IRF3, ITGA3,ITGA6, ITGB4, JAK1, JARID1A, JUN, JUNB, JUND, K-ALPHA-1, KIT, KITLG,KLK10, KPNA2, KRAS2, KRT18, KRT2A, KRT9, LAMB1, LAMP2, LCK, LCN2, LEP,LITAF, LRPAP1, LTF, LYN, LZTR1, MADH1, MAP2K2, MAP3K8, MAPK12, MAPK13,MAPKAPK3, MAPRE1, MARS, MAS1, MCC, MCM2, MCM4, MDM2, MDM4, MET, MGST1,MICB, MLLT3, MME, MMP1, MMP14, MMP17, MMP2, MNDA, MSH2, MSH6, MT3, MYB,MYBL1, MYBL2, MYC, MYCL1, MYCN, MYD88, MYL9, MYLK, NEO1, NF1, NF2,NFKB1, NFKB2, NFSF7, NID, NINJ1, NMBR, NME1, NME2, NME3, NOTCH1, NOTCH2,NOTCH4, NPM1, NQO1, NR1D1, NR2F1, NR2F6, NRAS, NRG1, NSEP1, OSM, PA2G4,PABPC1, PCNA, PCTK1, PCTK2, PCTK3, PDGFA, PDGFB, PDGFRA, PDPK1, PEA15,PFDN4, PFDN5, PGAM1, PHB, PIK3CA, PIK3CB, PIK3CG, PIM1, PKM2, PKMYT1,PLK2, PPARD, PPARG, PPIH, PPP1CA, PPP2R5A, PRDX2, PRDX4, PRKAR1A,PRKCBP1, PRNP, PRSS15, PSMA1, PTCH, PTEN, PTGS1, PTMA, PTN, PTPRN,RAB5A, RAC1, RAD50, RAF1, RALBP1, RAP1A, RARA, RARB, RASGRF1, RB1,RBBP4, RBL2, REA, REL, RELA, RELB, RET, RFC2, RGS19, RHOA, RHOB, RHOC,RHOD, RIPK1, RPN2, RPS6KB1, RRM1, SARS, SELENBP1, SEMA3C, SEMA4D, SEPP1,SERPINH1, SFN, SFPQ, SFRS7, SHB, SHH, SIAH2, SIVA, SIVA TP53, SKI, SKIL,SLC16A1, SLC1A4, SLC20A1, SMO, SMPD1, SNAI2, SND1, SNRPB2, SOCS1, SOCS3,SOD1, SORT1, SPINT2, SPRY2, SRC, SRPX, STAT1, STAT2, STAT3, STAT5B,STC1, TAF1, TBL3, TBRG4, TCF1, TCF7L2, TFAP2C, TFDP1, TFDP2, TGFA,TGFB1, TGFBI, TGFBR2, TGFBR3, THBS1, TIE, TIMP1, TIMP3, TJP1, TK1, TLE1,TNF, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF1B, TNFRSF6, TNFSF7, TNK1,TOB1, TP53, TP53BP2, TP53I3, TP73, TPBG, TPT1, TRADD, TRAM1, TRRAP,TSG101, TUFM, TXNRD1, TYRO3, UBC, UBE2L6, UCHL1, USP7, VDAC1, VEGF, VHL,VIL2, WEE1, WNT1, WNT2, WNT2B, WNT3, WNT5A, WT1, XRCC1, YES1, YWHAB,YWHAZ, ZAP70, and ZNF9.

In some embodiments, the instant disclosure relates to a heterologousnucleic acid insert encoding a gene product associated with aCNS-related disorder. The following is a non-limiting list of genesassociated with a CNS-related disorder: DRD2, GRIA1, GRIA2, GRIN1,SLC1A1, SYP, SYT1, CHRNA7, 3Rtau/4rTUS, APP, BAX, BCL-2, GRIK1, GFAP,IL-1, AGER, associated with Alzheimer's Disease; UCH-L1, SKP1, EGLN1,Nurr-1, BDNF, TrkB, gstm1, S106β, associated with Parkinson's Disease;IT15, PRNP, JPH3, TBP, ATXN1, ATXN2, ATXN3, Atrophin 1, FTL, TITF-1,associated with Huntington's Disease; FXN, associated with Freidrich'sataxia; ASPA, associated with Canavan's Disease; DMD, associated withmuscular dystrophy; and SMN1, UBE1, DYNC1H1 associated with spinalmuscular atrophy.

A heterologous nucleic acid insert may comprise as a transgene, anucleic acid encoding a protein or functional RNA that modulatesapoptosis. The following is a non-limiting list of genes associated withapoptosis and nucleic acids encoding the products of these genes andtheir homologues and encoding small interfering nucleic acids (e.g.,shRNAs, miRNAs) that inhibit the expression of these genes and theirhomologues are useful as transgenes in certain embodiments of thedisclosure: RPS27A, ABL1, AKT1, APAF1, BAD, BAG1, BAG3, BAG4, BAK1, BAX,BCL10, BCL2, BCL2A1, BCL2L1, BCL2L10, BCL2L11, BCL2L12, BCL2L13, BCL2L2,BCLAF1, BFAR, BID, BIK, NAIP, BIRC2, BIRC3, XIAP, BRCS, BIRC6, BIRC7,BIRC8, BNIP1, BNIP2, BNIP3, BNIP3L, BOK, BRAF, CARD10, CARD11, NLRC4,CARD14, NOD2, NOD1, CARD6, CARDS, CARDS, CASP1, CASP10, CASP14, CASP2,CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CFLAR, CIDEA, CIDEB,CRADD, DAPK1, DAPK2, DFFA, DFFB, FADD, GADD45A, GDNF, HRK, IGF1R, LTA,LTBR, MCL1, NOL3, PYCARD, RIPK1, RIPK2, TNF, TNFRSF10A, TNFRSF10B,TNFRSF10C, TNFRSF10D, TNFRSF11B, TNFRSF12A, TNFRSF14, TNFRSF19,TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF25, CD40, FAS, TNFRSF6B, CD27,TNFRSF9, TNFSF10, TNFSF14, TNFSF18, CD40LG, FASLG, CD70, TNFSF8, TNFSF9,TP53, TP53BP2, TP73, TP63, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5DRD2, GRIA1, GRIA2, GRIN1, SLC1A1, SYP, SYT1, CHRNA7, 3Rtau/4rTUS, APP,BAX, BCL-2, GRIK1, GFAP, IL-1, AGER, UCH-L1, SKP1, EGLN1, Nurr-1, BDNF,TrkB, gstm1, S106(3, IT15, PRNP, JPH3, TBP, ATXN1, ATXN2, ATXN3,Atrophin 1, FTL, TITF-1, FXN, ASPA, DMD, and SMN1, UBE1, DYNC1H1.

The skilled artisan will also realize that in the case of transgenesencoding proteins or polypeptides, that mutations that results inconservative amino acid substitutions may be made in a transgene toprovide functionally equivalent variants, or homologs of a protein orpolypeptide. In some aspects the disclosure embraces sequencealterations that result in conservative amino acid substitution of atransgene. In some embodiments, the transgene comprises a gene having adominant negative mutation. For example, a transgene may express amutant protein that interacts with the same elements as a wild-typeprotein, and thereby blocks some aspect of the function of the wild-typeprotein.

Useful transgene products also include miRNAs. miRNAs and other smallinterfering nucleic acids regulate gene expression via target RNAtranscript cleavage/degradation or translational repression of thetarget messenger RNA (mRNA). miRNAs are natively expressed, typically asfinal 19-25 non-translated RNA products. miRNAs exhibit their activitythrough sequence-specific interactions with the 3′ untranslated regions(UTR) of target mRNAs. These endogenously expressed miRNAs form hairpinprecursors which are subsequently processed into a miRNA duplex, andfurther into a “mature” single stranded miRNA molecule. This maturemiRNA guides a multiprotein complex, miRISC, which identifies targetsite, e.g., in the 3′ UTR regions, of target mRNAs based upon theircomplementarity to the mature miRNA.

The following non-limiting list of miRNA genes, and their homologues,are useful as transgenes or as targets for small interfering nucleicacids encoded by transgenes (e.g., miRNA sponges, antisenseoligonucleotides, TuD RNAs) in certain embodiments of the methods:hsa-let-7a, hsa-let-7a*, hsa-let-7b, hsa-let-7b*, hsa-let-7c,hsa-let-7c*, hsa-let-7d, hsa-let-7d*, hsa-let-7e, hsa-let-7e*,hsa-let-7f, hsa-let-7f-1*, hsa-let-7f-2*, hsa-let-7g, hsa-let-7g*,hsa-let-7i, hsa-let-7i*, hsa-miR-1, hsa-miR-100, hsa-miR-100*,hsa-miR-101, hsa-miR-101*, hsa-miR-103, hsa-miR-105, hsa-miR-105*,hsa-miR-106a, hsa-miR-106a*, hsa-miR-106b, hsa-miR-106b*, hsa-miR-107,hsa-miR-10a, hsa-miR-10a*, hsa-miR-10b, hsa-miR-10b*, hsa-miR-1178,hsa-miR-1179, hsa-miR-1180, hsa-miR-1181, hsa-miR-1182, hsa-miR-1183,hsa-miR-1184, hsa-miR-1185, hsa-miR-1197, hsa-miR-1200, hsa-miR-1201,hsa-miR-1202, hsa-miR-1203, hsa-miR-1204, hsa-miR-1205, hsa-miR-1206,hsa-miR-1207-3p, hsa-miR-1207-5p, hsa-miR-1208, hsa-miR-122,hsa-miR-122*, hsa-miR-1224-3p, hsa-miR-1224-5p, hsa-miR-1225-3p,hsa-miR-1225-5p, hsa-miR-1226, hsa-miR-1226*, hsa-miR-1227,hsa-miR-1228, hsa-miR-1228*, hsa-miR-1229, hsa-miR-1231, hsa-miR-1233,hsa-miR-1234, hsa-miR-1236, hsa-miR-1237, hsa-miR-1238, hsa-miR-124,hsa-miR-124*, hsa-miR-1243, hsa-miR-1244, hsa-miR-1245, hsa-miR-1246,hsa-miR-1247, hsa-miR-1248, hsa-miR-1249, hsa-miR-1250, hsa-miR-1251,hsa-miR-1252, hsa-miR-1253, hsa-miR-1254, hsa-miR-1255a, hsa-miR-1255b,hsa-miR-1256, hsa-miR-1257, hsa-miR-1258, hsa-miR-1259, hsa-miR-125a-3p,hsa-miR-125a-5p, hsa-miR-125b, hsa-miR-125b-1*, hsa-miR-125b-2*,hsa-miR-126, hsa-miR-126*, hsa-miR-1260, hsa-miR-1261, hsa-miR-1262,hsa-miR-1263, hsa-miR-1264, hsa-miR-1265, hsa-miR-1266, hsa-miR-1267,hsa-miR-1268, hsa-miR-1269, hsa-miR-1270, hsa-miR-1271, hsa-miR-1272,hsa-miR-1273, hsa-miR-127-3p, hsa-miR-1274a, hsa-miR-1274b,hsa-miR-1275, hsa-miR-127-5p, hsa-miR-1276, hsa-miR-1277, hsa-miR-1278,hsa-miR-1279, hsa-miR-128, hsa-miR-1280, hsa-miR-1281, hsa-miR-1282,hsa-miR-1283, hsa-miR-1284, hsa-miR-1285, hsa-miR-1286, hsa-miR-1287,hsa-miR-1288, hsa-miR-1289, hsa-miR-129*, hsa-miR-1290, hsa-miR-1291,hsa-miR-1292, hsa-miR-1293, hsa-miR-129-3p, hsa-miR-1294, hsa-miR-1295,hsa-miR-129-5p, hsa-miR-1296, hsa-miR-1297, hsa-miR-1298, hsa-miR-1299,hsa-miR-1300, hsa-miR-1301, hsa-miR-1302, hsa-miR-1303, hsa-miR-1304,hsa-miR-1305, hsa-miR-1306, hsa-miR-1307, hsa-miR-1308, hsa-miR-130a,hsa-miR-130a*, hsa-miR-130b, hsa-miR-130b*, hsa-miR-132, hsa-miR-132*,hsa-miR-1321, hsa-miR-1322, hsa-miR-1323, hsa-miR-1324, hsa-miR-133a,hsa-miR-133b, hsa-miR-134, hsa-miR-135a, hsa-miR-135a*, hsa-miR-135b,hsa-miR-135b*, hsa-miR-136, hsa-miR-136*, hsa-miR-137, hsa-miR-138,hsa-miR-138-1*, hsa-miR-138-2*, hsa-miR-139-3p, hsa-miR-139-5p,hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR-141, hsa-miR-141*,hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-143, hsa-miR-143*, hsa-miR-144,hsa-miR-144*, hsa-miR-145, hsa-miR-145*, hsa-miR-146a, hsa-miR-146a*,hsa-miR-146b-3p, hsa-miR-146b-5p, hsa-miR-147, hsa-miR-147b,hsa-miR-148a, hsa-miR-148a*, hsa-miR-148b, hsa-miR-148b*, hsa-miR-149,hsa-miR-149*, hsa-miR-150, hsa-miR-150*, hsa-miR-151-3p, hsa-miR-151-5p,hsa-miR-152, hsa-miR-153, hsa-miR-154, hsa-miR-154*, hsa-miR-155,hsa-miR-155*, hsa-miR-15a, hsa-miR-15a*, hsa-miR-15b, hsa-miR-15b*,hsa-miR-16, hsa-miR-16-1*, hsa-miR-16-2*, hsa-miR-17, hsa-miR-17*,hsa-miR-181a, hsa-miR-181a*, hsa-miR-181a-2*, hsa-miR-181b,hsa-miR-181c, hsa-miR-181c*, hsa-miR-181d, hsa-miR-182, hsa-miR-182*,hsa-miR-1825, hsa-miR-1826, hsa-miR-1827, hsa-miR-183, hsa-miR-183*,hsa-miR-184, hsa-miR-185, hsa-miR-185*, hsa-miR-186, hsa-miR-186*,hsa-miR-187, hsa-miR-187*, hsa-miR-188-3p, hsa-miR-188-5p, hsa-miR-18a,hsa-miR-18a*, hsa-miR-18b, hsa-miR-18b*, hsa-miR-190, hsa-miR-190b,hsa-miR-191, hsa-miR-191*, hsa-miR-192, hsa-miR-192*, hsa-miR-193a-3p,hsa-miR-193a-5p, hsa-miR-193b, hsa-miR-193b*, hsa-miR-194, hsa-miR-194*,hsa-miR-195, hsa-miR-195*, hsa-miR-196a, hsa-miR-196a*, hsa-miR-196b,hsa-miR-197, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-199a-5p,hsa-miR-199b-5p, hsa-miR-19a, hsa-miR-19a*, hsa-miR-19b, hsa-miR-19b-1*,hsa-miR-19b-2*, hsa-miR-200a, hsa-miR-200a*, hsa-miR-200b,hsa-miR-200b*, hsa-miR-200c, hsa-miR-200c*, hsa-miR-202, hsa-miR-202*,hsa-miR-203, hsa-miR-204, hsa-miR-205, hsa-miR-206, hsa-miR-208a,hsa-miR-208b, hsa-miR-20a, hsa-miR-20a*, hsa-miR-20b, hsa-miR-20b*,hsa-miR-21, hsa-miR-21*, hsa-miR-210, hsa-miR-211, hsa-miR-212,hsa-miR-214, hsa-miR-214*, hsa-miR-215, hsa-miR-216a, hsa-miR-216b,hsa-miR-217, hsa-miR-218, hsa-miR-218-1*, hsa-miR-218-2*,hsa-miR-219-1-3p, hsa-miR-219-2-3p, hsa-miR-219-5p, hsa-miR-22,hsa-miR-22*, hsa-miR-220a, hsa-miR-220b, hsa-miR-220c, hsa-miR-221,hsa-miR-221*, hsa-miR-222, hsa-miR-222*, hsa-miR-223, hsa-miR-223*,hsa-miR-224, hsa-miR-23a, hsa-miR-23a*, hsa-miR-23b, hsa-miR-23b*,hsa-miR-24, hsa-miR-24-1*, hsa-miR-24-2*, hsa-miR-25, hsa-miR-25*,hsa-miR-26a, hsa-miR-26a-1*, hsa-miR-26a-2*, hsa-miR-26b, hsa-miR-26b*,hsa-miR-27a, hsa-miR-27a*, hsa-miR-27b, hsa-miR-27b*, hsa-miR-28-3p,hsa-miR-28-5p, hsa-miR-296-3p, hsa-miR-296-5p, hsa-miR-297, hsa-miR-298,hsa-miR-299-3p, hsa-miR-299-5p, hsa-miR-29a, hsa-miR-29a*, hsa-miR-29b,hsa-miR-29b-1*, hsa-miR-29b-2*, hsa-miR-29c, hsa-miR-29c*, hsa-miR-300,hsa-miR-301a, hsa-miR-301b, hsa-miR-302a, hsa-miR-302a*, hsa-miR-302b,hsa-miR-302b*, hsa-miR-302c, hsa-miR-302c*, hsa-miR-302d, hsa-miR-302d*,hsa-miR-302e, hsa-miR-302f, hsa-miR-30a, hsa-miR-30a*, hsa-miR-30b,hsa-miR-30b*, hsa-miR-30c, hsa-miR-30c-1*, hsa-miR-30c-2*, hsa-miR-30d,hsa-miR-30d*, hsa-miR-30e, hsa-miR-30e*, hsa-miR-31, hsa-miR-31*,hsa-miR-32, hsa-miR-32*, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c,hsa-miR-320d, hsa-miR-323-3p, hsa-miR-323-5p, hsa-miR-324-3p,hsa-miR-324-5p, hsa-miR-325, hsa-miR-326, hsa-miR-328, hsa-miR-329,hsa-miR-330-3p, hsa-miR-330-5p, hsa-miR-331-3p, hsa-miR-331-5p,hsa-miR-335, hsa-miR-335*, hsa-miR-337-3p, hsa-miR-337-5p,hsa-miR-338-3p, hsa-miR-338-5p, hsa-miR-339-3p, hsa-miR-339-5p,hsa-miR-33a, hsa-miR-33a*, hsa-miR-33b, hsa-miR-33b*, hsa-miR-340,hsa-miR-340*, hsa-miR-342-3p, hsa-miR-342-5p, hsa-miR-345, hsa-miR-346,hsa-miR-34a, hsa-miR-34a*, hsa-miR-34b, hsa-miR-34b*, hsa-miR-34c-3p,hsa-miR-34c-5p, hsa-miR-361-3p, hsa-miR-361-5p, hsa-miR-362-3p,hsa-miR-362-5p, hsa-miR-363, hsa-miR-363*, hsa-miR-365, hsa-miR-367,hsa-miR-367*, hsa-miR-369-3p, hsa-miR-369-5p, hsa-miR-370,hsa-miR-371-3p, hsa-miR-371-5p, hsa-miR-372, hsa-miR-373, hsa-miR-373*,hsa-miR-374a, hsa-miR-374a*, hsa-miR-374b, hsa-miR-374b*, hsa-miR-375,hsa-miR-376a, hsa-miR-376a*, hsa-miR-376b, hsa-miR-376c, hsa-miR-377,hsa-miR-377*, hsa-miR-378, hsa-miR-378*, hsa-miR-379, hsa-miR-379*,hsa-miR-380, hsa-miR-380*, hsa-miR-381, hsa-miR-382, hsa-miR-383,hsa-miR-384, hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR-410, hsa-miR-411,hsa-miR-411*, hsa-miR-412, hsa-miR-421, hsa-miR-422a, hsa-miR-423-3p,hsa-miR-423-5p, hsa-miR-424, hsa-miR-424*, hsa-miR-425, hsa-miR-425*,hsa-miR-429, hsa-miR-431, hsa-miR-431*, hsa-miR-432, hsa-miR-432*,hsa-miR-433, hsa-miR-448, hsa-miR-449a, hsa-miR-449b, hsa-miR-450a,hsa-miR-450b-3p, hsa-miR-450b-5p, hsa-miR-451, hsa-miR-452,hsa-miR-452*, hsa-miR-453, hsa-miR-454, hsa-miR-454*, hsa-miR-455-3p,hsa-miR-455-5p, hsa-miR-483-3p, hsa-miR-483-5p, hsa-miR-484,hsa-miR-485-3p, hsa-miR-485-5p, hsa-miR-486-3p, hsa-miR-486-5p,hsa-miR-487a, hsa-miR-487b, hsa-miR-488, hsa-miR-488*, hsa-miR-489,hsa-miR-490-3p, hsa-miR-490-5p, hsa-miR-491-3p, hsa-miR-491-5p,hsa-miR-492, hsa-miR-493, hsa-miR-493*, hsa-miR-494, hsa-miR-495,hsa-miR-496, hsa-miR-497, hsa-miR-497*, hsa-miR-498, hsa-miR-499-3p,hsa-miR-499-5p, hsa-miR-500, hsa-miR-500*, hsa-miR-501-3p,hsa-miR-501-5p, hsa-miR-502-3p, hsa-miR-502-5p, hsa-miR-503,hsa-miR-504, hsa-miR-505, hsa-miR-505*, hsa-miR-506, hsa-miR-507,hsa-miR-508-3p, hsa-miR-508-5p, hsa-miR-509-3-5p, hsa-miR-509-3p,hsa-miR-509-5p, hsa-miR-510, hsa-miR-511, hsa-miR-512-3p,hsa-miR-512-5p, hsa-miR-513a-3p, hsa-miR-513a-5p, hsa-miR-513b,hsa-miR-513c, hsa-miR-514, hsa-miR-515-3p, hsa-miR-515-5p,hsa-miR-516a-3p, hsa-miR-516a-5p, hsa-miR-516b, hsa-miR-517*,hsa-miR-517a, hsa-miR-517b, hsa-miR-517c, hsa-miR-518a-3p,hsa-miR-518a-5p, hsa-miR-518b, hsa-miR-518c, hsa-miR-518c*,hsa-miR-518d-3p, hsa-miR-518d-5p, hsa-miR-518e, hsa-miR-518e*,hsa-miR-518f, hsa-miR-518f*, hsa-miR-519a, hsa-miR-519b-3p,hsa-miR-519c-3p, hsa-miR-519d, hsa-miR-519e, hsa-miR-519e*,hsa-miR-520a-3p, hsa-miR-520a-5p, hsa-miR-520b, hsa-miR-520c-3p,hsa-miR-520d-3p, hsa-miR-520d-5p, hsa-miR-520e, hsa-miR-520f,hsa-miR-520g, hsa-miR-520h, hsa-miR-521, hsa-miR-522, hsa-miR-523,hsa-miR-524-3p, hsa-miR-524-5p, hsa-miR-525-3p, hsa-miR-525-5p,hsa-miR-526b, hsa-miR-526b*, hsa-miR-532-3p, hsa-miR-532-5p,hsa-miR-539, hsa-miR-541, hsa-miR-541*, hsa-miR-542-3p, hsa-miR-542-5p,hsa-miR-543, hsa-miR-544, hsa-miR-545, hsa-miR-545*, hsa-miR-548a-3p,hsa-miR-548a-5p, hsa-miR-548b-3p, hsa-miR-548b-5p, hsa-miR-548c-3p,hsa-miR-548c-5p, hsa-miR-548d-3p, hsa-miR-548d-5p, hsa-miR-548e,hsa-miR-548f, hsa-miR-548g, hsa-miR-548h, hsa-miR-548i, hsa-miR-548j,hsa-miR-548k, hsa-miR-548l, hsa-miR-548m, hsa-miR-548n, hsa-miR-548o,hsa-miR-548p, hsa-miR-549, hsa-miR-550, hsa-miR-550*, hsa-miR-551a,hsa-miR-551b, hsa-miR-551b*, hsa-miR-552, hsa-miR-553, hsa-miR-554,hsa-miR-555, hsa-miR-556-3p, hsa-miR-556-5p, hsa-miR-557, hsa-miR-558,hsa-miR-559, hsa-miR-561, hsa-miR-562, hsa-miR-563, hsa-miR-564,hsa-miR-566, hsa-miR-567, hsa-miR-568, hsa-miR-569, hsa-miR-570,hsa-miR-571, hsa-miR-572, hsa-miR-573, hsa-miR-574-3p, hsa-miR-574-5p,hsa-miR-575, hsa-miR-576-3p, hsa-miR-576-5p, hsa-miR-577, hsa-miR-578,hsa-miR-579, hsa-miR-580, hsa-miR-581, hsa-miR-582-3p, hsa-miR-582-5p,hsa-miR-583, hsa-miR-584, hsa-miR-585, hsa-miR-586, hsa-miR-587,hsa-miR-588, hsa-miR-589, hsa-miR-589*, hsa-miR-590-3p, hsa-miR-590-5p,hsa-miR-591, hsa-miR-592, hsa-miR-593, hsa-miR-593*, hsa-miR-595,hsa-miR-596, hsa-miR-597, hsa-miR-598, hsa-miR-599, hsa-miR-600,hsa-miR-601, hsa-miR-602, hsa-miR-603, hsa-miR-604, hsa-miR-605,hsa-miR-606, hsa-miR-607, hsa-miR-608, hsa-miR-609, hsa-miR-610,hsa-miR-611, hsa-miR-612, hsa-miR-613, hsa-miR-614, hsa-miR-615-3p,hsa-miR-615-5p, hsa-miR-616, hsa-miR-616*, hsa-miR-617, hsa-miR-618,hsa-miR-619, hsa-miR-620, hsa-miR-621, hsa-miR-622, hsa-miR-623,hsa-miR-624, hsa-miR-624*, hsa-miR-625, hsa-miR-625*, hsa-miR-626,hsa-miR-627, hsa-miR-628-3p, hsa-miR-628-5p, hsa-miR-629, hsa-miR-629*,hsa-miR-630, hsa-miR-631, hsa-miR-632, hsa-miR-633, hsa-miR-634,hsa-miR-635, hsa-miR-636, hsa-miR-637, hsa-miR-638, hsa-miR-639,hsa-miR-640, hsa-miR-641, hsa-miR-642, hsa-miR-643, hsa-miR-644,hsa-miR-645, hsa-miR-646, hsa-miR-647, hsa-miR-648, hsa-miR-649,hsa-miR-650, hsa-miR-651, hsa-miR-652, hsa-miR-653, hsa-miR-654-3p,hsa-miR-654-5p, hsa-miR-655, hsa-miR-656, hsa-miR-657, hsa-miR-658,hsa-miR-659, hsa-miR-660, hsa-miR-661, hsa-miR-662, hsa-miR-663,hsa-miR-663b, hsa-miR-664, hsa-miR-664*, hsa-miR-665, hsa-miR-668,hsa-miR-671-3p, hsa-miR-671-5p, hsa-miR-675, hsa-miR-7, hsa-miR-708,hsa-miR-708*, hsa-miR-7-1*, hsa-miR-7-2*, hsa-miR-720, hsa-miR-744,hsa-miR-744*, hsa-miR-758, hsa-miR-760, hsa-miR-765, hsa-miR-766,hsa-miR-767-3p, hsa-miR-767-5p, hsa-miR-768-3p, hsa-miR-768-5p,hsa-miR-769-3p, hsa-miR-769-5p, hsa-miR-770-5p, hsa-miR-802,hsa-miR-873, hsa-miR-874, hsa-miR-875-3p, hsa-miR-875-5p,hsa-miR-876-3p, hsa-miR-876-5p, hsa-miR-877, hsa-miR-877*,hsa-miR-885-3p, hsa-miR-885-5p, hsa-miR-886-3p, hsa-miR-886-5p,hsa-miR-887, hsa-miR-888, hsa-miR-888*, hsa-miR-889, hsa-miR-890,hsa-miR-891a, hsa-miR-891b, hsa-miR-892a, hsa-miR-892b, hsa-miR-9,hsa-miR-9*, hsa-miR-920, hsa-miR-921, hsa-miR-922, hsa-miR-923,hsa-miR-924, hsa-miR-92a, hsa-miR-92a-1*, hsa-miR-92a-2*, hsa-miR-92b,hsa-miR-92b*, hsa-miR-93, hsa-miR-93*, hsa-miR-933, hsa-miR-934,hsa-miR-935, hsa-miR-936, hsa-miR-937, hsa-miR-938, hsa-miR-939,hsa-miR-940, hsa-miR-941, hsa-miR-942, hsa-miR-943, hsa-miR-944,hsa-miR-95, hsa-miR-96, hsa-miR-96*, hsa-miR-98, hsa-miR-99a,hsa-miR-99a*, hsa-miR-99b, and hsa-miR-99b*.

A miRNA inhibits the function of the mRNAs it targets and, as a result,inhibits expression of the polypeptides encoded by the mRNAs. Thus,blocking (partially or totally) the activity of the miRNA (e.g.,silencing the miRNA) can effectively induce, or restore, expression of apolypeptide whose expression is inhibited (derepress the polypeptide).In one embodiment, derepression of polypeptides encoded by mRNA targetsof a miRNA is accomplished by inhibiting the miRNA activity in cellsthrough any one of a variety of methods. For example, blocking theactivity of a miRNA can be accomplished by hybridization with a smallinterfering nucleic acid (e.g., antisense oligonucleotide, miRNA sponge,TuD RNA) that is complementary, or substantially complementary to, themiRNA, thereby blocking interaction of the miRNA with its target mRNA.As used herein, an small interfering nucleic acid that is substantiallycomplementary to a miRNA is one that is capable of hybridizing with amiRNA, and blocking the miRNA's activity. In some embodiments, an smallinterfering nucleic acid that is substantially complementary to a miRNAis an small interfering nucleic acid that is complementary with themiRNA at all but 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, or 18 bases. In some embodiments, an small interfering nucleic acidsequence that is substantially complementary to a miRNA, is an smallinterfering nucleic acid sequence that is complementary with the miRNAat, at least, one base.

A “miRNA Inhibitor” is an agent that blocks miRNA function, expressionand/or processing. For instance, these molecules include but are notlimited to microRNA specific antisense, microRNA sponges, tough decoyRNAs (TuD RNAs) and microRNA oligonucleotides (double-stranded, hairpin,short oligonucleotides) that inhibit miRNA interaction with a Droshacomplex. MicroRNA inhibitors can be expressed in cells from a transgenesof a nucleic acid, as discussed above. MicroRNA sponges specificallyinhibit miRNAs through a complementary heptameric seed sequence (Ebert,M. S. Nature Methods, Epub Aug. 12, 2007;). In some embodiments, anentire family of miRNAs can be silenced using a single sponge sequence.TuD RNAs achieve efficient and long-term-suppression of specific miRNAsin mammalian cells (See, e.g., Takeshi Haraguchi, et al., Nucleic AcidsResearch, 2009, Vol. 37, No. 6 e43, the contents of which relating toTuD RNAs are incorporated herein by reference). Other methods forsilencing miRNA function (derepression of miRNA targets) in cells willbe apparent to one of ordinary skill in the art.

In some aspects, nucleic acids described herein (e.g., ceDNA) may beuseful for the treatment of CNS-related disorders. As used herein, a“CNS-related disorder” is a disease or condition of the central nervoussystem. A CNS-related disorder may affect the spinal cord (e.g., amyelopathy), brain (e.g., a encephalopathy) or tissues surrounding thebrain and spinal cord. A CNS-related disorder may be of a geneticorigin, either inherited or acquired through a somatic mutation. ACNS-related disorder may be a psychological condition or disorder, e.g.,Attention Deficient Hyperactivity Disorder, Autism Spectrum Disorder,Mood Disorder, Schizophrenia, Depression, Rhett Syndrome, etc. ACNS-related disorder may be an autoimmune disorder. A CNS-relateddisorder may also be a cancer of the CNS, e.g., brain cancer. ACNS-related disorder that is a cancer may be a primary cancer of theCNS, e.g., an astrocytoma, glioblastomas, etc., or may be a cancer thathas metastasized to CNS tissue, e.g., a lung cancer that hasmetastasized to the brain. Further non-limiting examples of CNS-relateddisorders, include Parkinson's Disease, Lysosomal Storage Disease,Ischemia, Neuropathic Pain, Amyotrophic lateral sclerosis (ALS),Multiple Sclerosis (MS), and Canavan disease (CD).

In some embodiments, nucleic acids (e.g., ceDNA) described herein may beuseful for delivering gene therapy to cardiac cells (e.g., hearttissue). Accordingly, in some embodiments, nucleic acids (e.g., ceDNA)described herein may be useful for the treatment of cardiovasculardisorders. As used herein, a “cardiovascular disorder” is a disease orcondition of the cardiovascular system. A cardiovascular disease mayaffect the heart, circulatory system, arteries, veins, blood vesselsand/or capillaries. A cardiovascular disorder may be of a geneticorigin, either inherited or acquired through a somatic mutation.Non-limiting examples of cardiovascular disorders include rheumaticheart disease, valvular heart disease, hypertensive heart disease,aneurysm, atherosclerosis, hypertension (e.g., high blood pressure),peripheral arterial disease (PAD), ischemic heart disease, angina,coronary heart disease, coronary artery disease, myocardial infarction,cerebral vascular disease, transient ischemic attack, inflammatory heartdisease, cardiomyopathy, pericardial disease, congenital heart disease,heart failure, stroke, and myocarditis due to Chagas disease.

In some embodiments, nucleic acids described herein (e.g., ceDNA) maytarget the lung and/or tissue of the pulmonary system. Accordingly, insome embodiments, nucleic acids (e.g., ceDNA) described herein may beuseful for treatment of pulmonary disease. As used herein a “pulmonarydisease” is a disease or condition of the pulmonary system. A pulmonarydisease may affect the lungs or muscles involved in breathing. Apulmonary disease may be of a genetic origin, either inherited oracquired through a somatic mutation. A pulmonary disease may be a cancerof the lung, including but not limited to, non-small cell lung cancer,small cell lung cancer, and lung carcinoid tumor. Further non-limitingexamples of pulmonary diseases include acute bronchitis, acuterespiratory distress syndrome (ARDS), asbestosis, asthma,bronchiectasis, bronchiolitis, bronchiolitis obliterans organizingpneumonia (BOOP), bronchopulmonary dysplasia, byssinosis, chronicbronchitis, coccidioidomycosis (Cocci), chronic obstructive pulmonarydisorder (COPD), cryptogenic organizing pneumonia (COP), cysticfibrosis, emphysema, Hantavirus Pulmonary Syndrome, histoplasmosis,Human Metapneumovirus, hypersensitivity pneumonitis, influenza,lymphangiomatosis, mesothelioma, Middle Eastern Respiratory Syndrome,non-tuberculosis Mycobacterium, Pertussis, Pneumoconiosis (Black LungDisease), pneumonia, primary ciliary dyskinesia, primary pulmonaryhypertension, pulmonary arterial hypertension, pulmonary fibrosis,pulmonary vascular disease, Respiratory Syncytial Virus (RSV),sarcoidosis, Severe Acute Respiratory Syndrome (SARS), silicosis, sleepapnea, Sudden Infant Death Syndrome (SIDS), and tuberculosis.

In some embodiments, nucleic acids described herein (e.g., ceDNA) maytarget liver tissue. Accordingly, in some embodiments, nucleic acidsdescribed herein (e.g., ceDNA) may be useful for treatment of hepaticdisease. As used herein a “hepatic disease” is a disease or condition ofthe liver. A hepatic disease may be of a genetic origin, eitherinherited or acquired through a somatic mutation. A hepatic disease maybe a cancer of the liver, including but not limited to hepatocellularcarcinoma (HCC), fibrolamellar carcinoma, cholangiocarcinoma,angiosarcoma and hepatoblastoma. Further non-limiting examples ofpulmonary diseases include Alagille Syndrome, Alpha 1 Anti-TrypsinDeficiency, autoimmune hepatitis, biliary atresia, cirrhosis, cysticdisease of the liver, fatty liver disease, galactosemia, gallstones,Gilbert's Syndrome, hemochromatosis, liver disease in pregnancy,neonatal hepatitis, primary biliary cirrhosis, primary sclerosingcholangitis, porphyria, Reye's Syndrome, sarcoidosis, toxic hepatitis,Type 1 Glycogen Storage Disease, tyrosinemia, viral hepatitis A, B, C,Wilson Disease, and schistosomiasis.

In some embodiments, nucleic acids described herein (e.g., ceDNA) maytarget kidney tissue. Accordingly, in some embodiments, nucleic acidsdescribed herein (e.g., ceDNA) may be useful for treatment of kidneydisease. As used herein a “kidney disease” is a disease or condition ofthe liver. A hepatic disease may be of a genetic origin, eitherinherited or acquired through a somatic mutation. A hepatic disease maybe a cancer of the kidney, including but not limited to renal cellcancer, clear cell cancer, papillary cancer type 1, papillary cancertype 2, chromophobe cancer, oncocytic cell cancer, collecting ductcancer, transitional cell cancer of the renal pelvis and Wilm's tumor.Further non-limiting examples of kidney disease includeAbderhalden-Kaufmann-Lignac syndrome (Nephropathic Cystinosis), AcuteKidney Failure/Acute Kidney Injury, Acute Lobar Nephronia, AcutePhosphate Nephropathy, Acute Tubular Necrosis, AdeninePhosphoribosyltransferase Deficiency, Adenovirus Nephritis, AlportSyndrome, Amyloidosis, Angiomyolipoma, Analgesic Nephropathy,Angiotensin Antibodies and Focal Segmental Glomerulosclerosis,Antiphospholipid Syndrome, Anti-TNF-α Therapy-relatedGlomerulonephritis, APOL1 Mutations, Apparent Mneralocorticoid ExcessSyndrome, Aristolochic Acid Nephropathy, Balkan Endemic Nephropathy,Bartter Syndrome, Beeturia, f3-Thalassemia Renal Disease, Bile CastNephropathy, BK Polyoma, C1q Nephropathy, Cardiorenal syndrome, CFHR5nephropathy, Cholesterol Emboli, Churg-Strauss syndrome, Chyluria,Collapsing Glomerulopathy, Collapsing Glomerulopathy Related to CMV,Congenital Nephrotic Syndrome, Conorenal syndrome (Mainzer-SaldinoSyndrome or Saldino-Mainzer Disease), Contrast Nephropathy, CopperSulfate Intoxication, Cortical Necrosis, Cryoglobuinemia,Crystal-Induced Acute Kidney injury, Cystic Kidney Disease, Acquired,Cystinuria, Dense Deposit Disease (MPGN Type 2), Dent Disease (X-linkedRecessive Nephrolithiasis), Dialysis Disequilibrium Syndrome, DiabeticKidney Disease, Diabetes Insipidus, EAST syndrome, Ectopic Ureter,Edema, Erdheim-Chester Disease, Fabry's Disease, Familial HypocalciuricHypercalcemia, Fanconi Syndrome, Fraser syndrome, FibronectinGlomerulopathy, Fibrillary Glomerulonephritis and ImmunotactoidGlomerulopathy, Fraley syndrome, Focal Segmental Glomerulosclerosis,Focal Sclerosis, Focal Glomerulosclerosis, Galloway Mowat syndrome,Gitelman Syndrome, Glomerular Diseases, Glomerular Tubular Reflux,Glycosuria, Goodpasture Syndrome, Hemolytic Uremic Syndrome (HUS),Atypical Hemolytic Uremic Syndrome (aHUS), Hemophagocytic Syndrome,Hemorrhagic Cystitis, Hemosiderosis related to Paroxysmal NocturnalHemoglobinuria and Hemolytic Anemia, Hepatic Veno-Occlusive Disease,Sinusoidal Obstruction Syndrome, Hepatitis C-Associated Renal Disease,Hepatorenal Syndrome, HIV-Associated Nephropathy (HIVAN), HorseshoeKidney (Renal Fusion), Hunner's Ulcer, Hyperaldosteronism,Hypercalcemia, Hyperkalemia, Hypermagnesemia, Hypernatremia,Hyperoxaluria, Hyperphosphatemia, Hypocalcemia, Hypokalemia,Hypokalemia-induced renal dysfunction, Hypomagnesemia, Hyponatremia,Hypophosphatemia, IgA Nephropathy, IgG4 Nephropathy, InterstitialCystitis, Painful Bladder Syndrome, Interstitial Nephritis, Ivemark'ssyndrome, Kidney Stones, Nephrolithiasis, Leptospirosis Renal Disease,Light Chain Deposition Disease, Monoclonal Immunoglobulin DepositionDisease, Liddle Syndrome, Lightwood-Albright Syndrome, LipoproteinGlomerulopathy, Lithium Nephrotoxicity, LMX1B Mutations Cause HereditaryFSGS, Loin Pain Hematuria, Lupus, Systemic Lupus Erythematosis, LupusKidney Disease, Lupus Nephritis, Lyme Disease-AssociatedGlomerulonephritis, Malarial Nephropathy, Malignant Hypertension,Malakoplakia, Meatal Stenosis, Medullary Cystic Kidney Disease,Medullary Sponge Kidney, Megaureter, Melamine Toxicity and the Kidney,Membranoproliferative Glomerulonephritis, Membranous Nephropathy,MesoAmerican Nephropathy, Metabolic Acidosis, Metabolic Alkalosis,Microscopic Polyangiitis, Milk-alkalai syndrome, Minimal Change Disease,Multicystic dysplastic kidney, Multiple Myeloma, MyeloproliferativeNeoplasms and Glomerulopathy, Nail-patella Syndrome, Nephrocalcinosis,Nephrogenic Systemic Fibrosis, Nephroptosis (Floating Kidney, RenalPtosis), Nephrotic Syndrome, Neurogenic Bladder, NodularGlomerulosclerosis, Non-Gonococcal, Nutcracker syndrome, OrofaciodigitalSyndrome, Orthostatic Hypotension, Orthostatic Proteinuria, OsmoticDiuresis, Page Kidney, Papillary Necrosis, Papillorenal Syndrome(Renal-Coloboma Syndrome, Isolated Renal Hypoplasia), ThePeritoneal-Renal Syndrome, Posterior Urethral Valve, Post-infectiousGlomerulonephritis, Post-streptococcal Glomerulonephritis, PolyarteritisNodosa, Polycystic Kidney Disease, Posterior Urethral Valves,Preeclampsia, Proliferative Glomerulonephritis with Monoclonal IgGDeposits (Nasr Disease), Proteinuria (Protein in Urine),Pseudohyperaldosteronism, Pseudohypoparathyroidism, Pulmonary-RenalSyndrome, Pyelonephritis (Kidney Infection), Pyonephrosis, RadiationNephropathy, Refeeding syndrome, Reflux Nephropathy, Rapidly ProgressiveGlomerulonephritis, Renal Abscess, Peripnephric Abscess, Renal Agenesis,Renal Artery Aneurysm, Renal Artery Stenosis, Renal Cell Cancer, RenalCyst, Renal Hypouricemia with Exercise-induced Acute Renal Failure,Renal Infarction, Renal Osteodystrophy, Renal Tubular Acidosis, ResetOsmostat, Retrocaval Ureter, Retroperitoneal Fibrosis, Rhabdomyolysis,Rhabdomyolysis related to Bariatric Sugery, RheumatoidArthritis-Associated Renal Disease, Sarcoidosis Renal Disease, SaltWasting, Renal and Cerebral, Schimke immuno-osseous dysplasia,Scleroderma Renal Crisis, Serpentine Fibula-Polycystic Kidney Syndrome,Exner Syndrome, Sickle Cell Nephropathy, Silica Exposure and ChronicKidney Disease, Kidney Disease Following Hematopoietic CellTransplantation, Kidney Disease Related to Stem Cell Transplantation,Thin Basement Membrane Disease, Benign Familial Hematuria, Trigonitis,Tuberous Sclerosis, Tubular Dysgenesis, Tumor Lysis Syndrome, Uremia,Uremic Optic Neuropathy, Ureterocele, Urethral Caruncle, UrethralStricture, Urinary Incontinence, Urinary Tract Infection, Urinary TractObstruction, Vesicointestinal Fistula, Vesicoureteral Reflux, VonHippel-Lindau Disease, Warfarin-Related Nephropathy, Wegener'sGranulomatosis, Granulomatosis with Polyangiitis, and Wunderlichsyndrome.

In some embodiments, nucleic acids described herein (e.g., ceDNA) may beuseful for delivering gene therapy to ocular tissue. Accordingly, insome embodiments, nucleic acids described herein (e.g., ceDNA) may beuseful for the treatment of ocular disorders. As used herein, an “oculardisorder” is a disease or condition of the eye. A cardiovascular diseasemay affect the eye, sclera, cornea, anterior chamber, posterior chamber,iris, pupil, lens, vitreous humor, retina, or optic nerve. An oculardisorder may be of a genetic origin, either inherited or acquiredthrough a somatic mutation. Non-limiting examples of ocular diseases anddisorders include but are not limited to: age-related maculardegeneration, retinopathy, diabetic retinopathy, macula edema, glaucoma,retinitis pigmentosa, Stargardt's disease, Usher's disease and Leber'scongenital amaurosis and eye cancer.

In some embodiments, nucleic acids described herein (e.g., ceDNA) may beuseful for delivering gene therapy to blood tissue (e.g., blood cells).Accordingly, in some embodiments, nucleic acids described herein (e.g.,ceDNA) may be useful for the treatment of blood disorders. As usedherein, a “blood disorder” is a disease or condition of the blood. Ablood disorder may be of a genetic origin, either inherited or acquiredthrough a somatic mutation. Non-limiting examples of blood diseases anddisorders include but are not limited to anemia (e.g., anemia in chronickidney disease, aplastic anemia, myelodysplastic anemia, sickle cellanemia), deep vein thrombosis, hemophilia (e.g., hemophilia A,hemophilia B, hemophilia C), Henoch-Schönlein Purpura, pulmonaryembolism, thalassemia, and Von Willebrand disease.

In some embodiments, nucleic acids described herein (e.g., ceDNA) may beuseful for delivering gene editing molecules (e.g., nucleases) to asubject. In some embodiments a nucleic acid described by the disclosurecomprises a heterologous nucleic acid insert encodes a nuclease. As usedherein, the terms “endonuclease” and “nuclease” refer to an enzyme thatcleaves a phosphodiester bond or bonds within a polynucleotide chain.Nucleases may be naturally occurring or genetically engineered.Genetically engineered nucleases are particularly useful for genomeediting and are generally classified into four families: zinc fingernucleases (ZFNs), transcription activator-like effector nucleases(TALENs), engineered meganucleases and CRISPR-associated proteins (Casnucleases). In some embodiments, the nuclease is a ZFN. In someembodiments, the ZFN comprises a Fokl cleavage domain. In someembodiments, the ZFN comprises Cys2His2 fold group. In some embodiments,the nuclease is a TALEN. In some embodiments, the TALEN comprises a Foklcleavage domain. In some embodiments, the nuclease is an engineeredmeganuclease.

The term “CRISPR” refers to “clustered regularly interspaced shortpalindromic repeats”, which are DNA loci containing short repetitions ofbase sequences. CRISPR loci form a portion of a prokaryotic adaptiveimmune system that confers resistance to foreign genetic material. EachCRISPR loci is flanked by short segments of “spacer DNA”, which arederived from viral genomic material. In the Type II CRISPR system,spacer DNA hybridizes to transactivating RNA (tracrRNA) and is processedinto CRISPR-RNA (crRNA) and subsequently associates withCRISPR-associated nucleases (Cas nucleases) to form complexes thatrecognize and degrade foreign DNA. In certain embodiments, the nucleaseis a CRISPR-associated nuclease (Cas nuclease). Examples of CRISPRnucleases include, but are not limited to Cas9, Cas6 and dCas9. dCas9 isan engineered Cas protein that binds to a target locus but does notcleave said locus. In some embodiments, the nuclease is Cas9. In someembodiments, the Cas9 is derived from the bacteria S. pyogenes (SpCas9).

For the purpose of genome editing, the CRISPR system can be modified tocombine the tracrRNA and crRNA in to a single guide RNA (sgRNA) or just(gRNA). As used herein, the term “guide RNA” or “gRNA” refers to apolynucleotide sequence that is complementary to a target sequence in acell and associates with a Cas nuclease, thereby directing the Casnuclease to the target sequence. In some embodiments, a nucleic aciddescribed by the disclosure comprises a heterologous nucleic acid insertencoding a guide RNA (gRNA). In some embodiments, a gRNA ranges between1 and 30 nucleotides in length. In some embodiments, a gRNA rangesbetween 5 and 25 nucleotides in length. In some embodiments, a gRNAranges between 10 and 20 nucleotides in length. In some embodiments, agRNA ranges between 14 and 18 nucleotides in length. In someembodiments, a nucleic acid described by the disclosure comprises aheterologous nucleic acid insert encoding a gRNA and a CRISPR nuclease.

In some aspects, the disclosure relates to a nucleic acid encoding aheterologous nucleic acid insert that does not encode a functionalprotein. For example, in the context of gene therapy, transgene promoterintegration may cause oncogene activation. Accordingly, in someembodiments, the disclosure relates to a heterologous nucleic acidinsert encoding a promoterless construct. Without wishing to be bound byany particular theory, a promoterless expression construct is useful, insome embodiments, as a substrate for gene editing.

As used herein, “genome editing” refers to adding, disrupting orchanging genomic sequences (e.g., a gene sequence). In some embodiments,genome editing is performed using engineered proteins and relatedmolecules. In some aspects, genome editing comprises the use ofengineered nucleases to cleave a target genomic locus. In someembodiments, genome editing further comprises inserting, deleting,mutating or substituting nucleic acid residues at a cleaved locus. Insome embodiments, inserting, deleting, mutating or substituting nucleicacid residues at a cleaved locus is accomplished through endogenouscellular mechanisms such as homologous recombination (HR) andnon-homologous end joining (NHEJ). Exemplary genome editing technologiesinclude, but are not limited to Transcription Activator-like EffectorNucleases (TALENs), Zinc Finger Nucleases (ZFNs), engineeredmeganuclease re-engineered homing endonucleases, the CRISPR/Cas system.In some embodiments, the gene editing technologies are proteins ormolecules related to TALENs, including but not limited to transcriptionactivator-like effectors (TALEs) and restriction endonucleases (e.g.,Fokl). In some embodiments, the gene editing technologies are proteinsor molecules related to ZFNs, including but not limited to proteinscomprising the Cys2His2 fold group (for example Zif268 (EGR1)), andrestriction endonucleases (e.g., Fokl). In some embodiments, the geneediting technologies are proteins or molecules related to the CRISPR/Cassystem, including but not limited to Cas9, Cas6, dCas9, CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). In some embodiments, thepromoterless construct provides a substrate for TALENS, zinc fingernucleases (ZFNs), meganucleases, Cas9, and other gene editing proteins.

In some aspects, the disclosure relates to a nucleic acid encoding aheterologous nucleic acid insert that encodes a DNA vaccine. As usedherein, “DNA vaccine” refers to a nucleic acid encoding an antigen thatstimulates an immune response (e.g., a cellular immune response or ahumoral immune response) against that antigen in a host. In someembodiments, the immune response is a protective response that protectsagainst a future infection or condition. However, in some embodiments,the immune response treats (e.g., eradicates or attenuates) an existinginfection or condition. Examples of DNA vaccines include HL chain Ig,scFv, single-domain Ig derived from camelidae (VhH) or cartilaginousfish (Vnar), nanobody, and other paratope recognitions peptides andfusion peptides collectively referred to as Ig (or Ig-like) molecules.

In some embodiments, a heterologous nucleic acid insert encodes an Ig orIg-like molecule. In some embodiments, the Ig (or Ig-like) molecules areunmodified protein sequences derived from monoclonal antibody sequences.In some embodiments, the Ig (or Ig-like) molecules are unmodifiedprotein sequences derived from murine or other mammalian monoclonalantibody sequences.

In some embodiments, the Ig (or Ig-like) molecules are unmodifiedprotein sequences derived from synthetic randomly generated peptidelibraries. In some embodiments, the libraries were derived fromcomplimentary DNA obtained from naïve vertebrate species. The speciesinclude, but are not limited to mammals, such as primates (e.g., humansand non-human primates), rodents (e.g., mouse, rats), ungulates,camelids, equines, canines, felines, marsupials, and animals ofagricultural interest; Avian species, including chickens, ducks, andgeese; piscine species including cartlilaginous fish, lamprey eels, andjawed fish species.

In some embodiments, the heterologous nucleic acid encodes the heavy andlight chains for an immunoglobulin (Ig), such that when administered toa permissive cell, an assembled Ig is secreted into the circulatorysystem. In some embodiments, the Ig molecule is not secreted and actsinternally as a so-called “intra-body”.

In some embodiments, a heterologous nucleic acid insert encodes an Igmolecule that is an engineered single-chain antibody consisting of theheavy and light chain variable regions in one polypeptide (scFv). ThescFv retains avidity and specificity for the target antigen.

In some embodiments, a heterologous nucleic acid insert encodes an Igmolecule that binds to microbial agents and affects the infectivity ofthe microbe. In some embodiments, the microbial agents are prokaryoticorganisms. In some embodiments, the microbial agents are rickettsia,mycoplasma, or other intracellular life forms.

In some embodiments, a heterologous nucleic acid insert encodes an Igmolecule that binds to viral structural protein(s) of human pathogenicviruses, including but not limited an Ebola virus viral protein, a humanimmune deficiency viral protein, a papilloma viral protein, a herpessimplex 1 viral protein, a herpes simplex 2 viral protein, a HCV A viralprotein, a HCV B viral protein, a HCV C viral protein, a HCV non-A viralprotein, a HCV non-B viral protein, or a dengue hemorrhagic fever viralprotein. In some embodiments, a heterologous nucleic acid insert encodesan Ig molecule that binds to viral structural protein(s) of a zoonoticpathogen, including, but not limited to foot and mouth disease virus andrabies virus.

In some aspects, nucleic acids described by the disclosure are usefulfor the production of modified cells, such as ex vivo modified cells. Asused herein, “ex vivo modified cell” refers to a cell (e.g., a mammaliancell) that is removed from a subject, genetically modified (e.g.,transfected or transduced with exogenous nucleic acids, or geneticallyreprogrammed), cultured or expanded, and optionally, returned to asubject (e.g., either the same subject, or a different subject).Generally, ex vivo modified cells are useful for autologous celltherapy, or allogeneic cell therapy. For example, cells may be removedfrom a subject having a disease associated with a particular geneticdefect (e.g., overexpression of a particular protein), transfected witha nucleic acid that corrects the genetic defect (e.g. reduces expressionof the protein), and reintroduced into the subject. In anothernon-limiting example, cells are removed from a subject, geneticallyreprogrammed (e.g., dedifferentiated or transdifferentiated into stemcells), expanded, and reintroduced into the subject. In someembodiments, ex vivo modified cells produced by transfection with anucleic acid as described by the disclosure have an improved safetyprofile compared to ex vivo cells produced by currently available genetherapy vectors.

In some aspects, nucleic acids described by the disclosure are usefulfor the production of chimeric antigen T-cells (CARTs). Chimeric AntigenReceptors (CARs) are engineered T cell receptors displaying specificityagainst target antigens based on a single chain FV (scFv) antibodymoiety. Generally, CARTs are produced by transduction of T-cells withlentiviral vectors comprising DNA encoding CARs. Lentiviraltransduction, in some embodiments, raises the risk of insertionalmutagenesis leading to cancer. As described by the disclosure, nucleicacids having asymmetric interrupted self-complementary sequences exhibitreduced likelihood of insertional mutagenesis compared to other genetherapy modalities. Accordingly, in some embodiments a nucleic aciddescribed by the disclosure comprises a heterologous nucleic acid insert(e.g., a transgene) encoding a CAR. In some embodiments, a CART producedby transduction with a nucleic acid described by the disclosure exhibitsan improved safety profile compared to a CART produced by lentiviraltransduction.

Additional Components

In addition to the major elements identified above for the nucleic acid,the nucleic acid also includes conventional control elements necessarywhich are operably linked to the heterologous nucleic acid insert (e.g.,transgene) in a manner which permits its transcription, translationand/or expression in a cell transfected with a nucleic acid described bythe disclosure. As used herein, “operably linked” sequences include bothexpression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance to control the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters which are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

In some embodiments, a heterologous nucleic acid insert (e.g.,transgene) comprises a protein coding sequence that is operably linkedto one or more regulatory sequences. As used herein, a nucleic acidcoding sequence and regulatory sequences are said to be “operably”linked when they are covalently linked in such a way as to place theexpression or transcription of the nucleic acid sequence (e.g.,transgene) under the influence or control of the regulatory sequences.If it is desired that the nucleic acid sequence (e.g., transgene) betranslated into a functional protein, two DNA sequences are said to beoperably linked if induction of a promoter in the 5′ regulatorysequences results in the transcription of the coding sequence and if thenature of the linkage between the two DNA sequences does not (1) resultin the introduction of a frame-shift mutation, (2) interfere with theability of the promoter region to direct the transcription of the codingsequences, or (3) interfere with the ability of the corresponding RNAtranscript to be translated into a protein. Thus, a promoter regionwould be operably linked to a nucleic acid sequence if the promoterregion were capable of effecting transcription of that DNA sequence suchthat the resulting transcript might be translated into the desiredprotein or polypeptide. Similarly two or more coding regions areoperably linked when they are linked in such a way that theirtranscription from a common promoter results in the expression of two ormore proteins having been translated in frame. In some embodiments,operably linked coding sequences yield a fusion protein. In someembodiments, operably linked coding sequences yield a functional RNA(e.g., gRNA).

For nucleic acids (e.g., transgenes) encoding proteins, apolyadenylation sequence generally is inserted following the transgenesequences and before the 3′ AAV ITR sequence. A heterologous nucleicacid insert (e.g., transgene) useful in the present disclosure may alsocontain an intron, desirably located between the promoter/enhancersequence and the transgene. One possible intron sequence is derived fromSV-40, and is referred to as the SV-40 T intron sequence. Another vectorelement that may be used is an internal ribosome entry site (IRES). AnIRES sequence is used to produce more than one polypeptide from a singlegene transcript. An IRES sequence would be used to produce a proteinthat contain more than one polypeptide chains. Selection of these andother common vector elements are conventional and many such sequencesare available [see, e.g., Sambrook et al, and references cited thereinat, for example, pages 3.18 3.26 and 16.17 16.27 and Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, New York,1989]. In some embodiments, a Foot and Mouth Disease Virus 2A sequenceis included in polyprotein; this is a small peptide (approximately 18amino acids in length) that has been shown to mediate the cleavage ofpolyproteins (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M etal., J Virology, November 1996; p. 8124-8127; Furler, S et al., GeneTherapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal,1999; 4: 453-459). The cleavage activity of the 2A sequence haspreviously been demonstrated in artificial systems including plasmidsand gene therapy vectors (AAV and retroviruses) (Ryan, M D et al., EMBO,1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996; p.8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin,C et al., The Plant Journal, 1999; 4: 453-459; de Felipe, P et al., GeneTherapy, 1999; 6: 198-208; de Felipe, P et al., Human Gene Therapy,2000; 11: 1921-1931; and Klump, H et al., Gene Therapy, 2001; 8:811-817).

The precise nature of the regulatory sequences needed for geneexpression in host cells may vary between species, tissues or celltypes, but shall in general include, as necessary, 5′ non-transcribedand 5′ non-translated sequences involved with the initiation oftranscription and translation respectively, such as a TATA box, cappingsequence, CAAT sequence, enhancer elements, and the like. Especially,such 5′ non-transcribed regulatory sequences will include a promoterregion that includes a promoter sequence for transcriptional control ofthe operably joined gene. Regulatory sequences may also include enhancersequences or upstream activator sequences as desired. The vectors of thedisclosure may optionally include 5′ leader or signal sequences. Thechoice and design of an appropriate vector is within the ability anddiscretion of one of ordinary skill in the art.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], theSV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter [Invitrogen].

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art.Examples of inducible promoters regulated by exogenously suppliedpromoters include the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); theecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA,93:3346-3351 (1996)), the tetracycline-repressible system (Gossen etal., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), thetetracycline-inducible system (Gossen et al., Science, 268:1766-1769(1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518(1998)), the RU486-inducible system (Wang et al., Nat. Biotech.,15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) and therapamycin-inducible system (Magari et al., J. Clin. Invest.,100:2865-2872 (1997)). Still other types of inducible promoters whichmay be useful in this context are those which are regulated by aspecific physiological state, e.g., temperature, acute phase, aparticular differentiation state of the cell, or in replicating cellsonly.

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

In some embodiments, the regulatory sequences impart tissue-specificgene expression capabilities. In some cases, the tissue-specificregulatory sequences bind tissue-specific transcription factors thatinduce transcription in a tissue specific manner. Such tissue-specificregulatory sequences (e.g., promoters, enhancers, etc.) are well knownin the art. Exemplary tissue-specific regulatory sequences include, butare not limited to the following tissue specific promoters: aliver-specific thyroxin binding globulin (TBG) promoter, an insulinpromoter, a glucagon promoter, a somatostatin promoter, a pancreaticpolypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatinekinase (MCK) promoter, a mammalian desmin (DES) promoter, a α-myosinheavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT) promoter.Other exemplary promoters include Beta-actin promoter, hepatitis B viruscore promoter, Sandig et al., Gene Ther., 3:1002-9 (1996);alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther.,7:1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol. Biol.Rep., 24:185-96 (1997)); bone sialoprotein promoter (Chen et al., J.Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J.Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cellreceptor α-chain promoter, neuronal such as neuron-specific enolase(NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15(1993)), neurofilament light-chain gene promoter (Piccioli et al., Proc.Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgfgene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), among otherswhich will be apparent to the skilled artisan.

Production of Closed-Ended Linear Duplex DNA (ceDNA)

In some aspects, the disclosure provides a method of producing a nucleicacid as described by the disclosure (e.g., ceDNA), comprising: (i)introducing into a permissive cell a nucleic acid encoding aheterologous nucleic acid insert flanked by at least one interruptedself-complementary sequence, each self-complementary sequence having anoperative terminal resolution site and a rolling circle replicationprotein binding element, wherein the self-complementary sequence isinterrupted by a cross-arm sequence forming two opposing,lengthwise-symmetric stem-loops, each of the opposinglengthwise-symmetric stem-loops having a stem portion in the range of 5to 15 base pairs in length and a loop portion having 2 to 5 unpaireddeoxyribonucleotides; and, (ii) maintaining the permissive cell underconditions in which a rolling circle replication protein in thepermissive cell initiates production of multiple copies of the nucleicacid.

The number of copies resulting from production of a nucleic acid can beexpressed as a multiple of the original number of copies of the nucleicacid (e.g., 1, 2, 10, 100, or more original copies) introduced into thepermissive cell. In some embodiments, production of multiple copies ofthe nucleic acid results in between 2-fold and 10,000-fold increase inthe number of copies of the nucleic acid in the permissive cell. In someembodiments, production of multiple copies of the nucleic acid resultsin greater than 10,000-fold increase in the number of copies of thenucleic acid in the permissive cell.

In some aspects, the disclosure provides transfected cells (e.g.,transfected permissive cells). The term “transfection” is used to referto the uptake of foreign DNA by a cell, and a cell has been“transfected” when exogenous DNA has been introduced inside the cellmembrane. A number of transfection techniques are generally known in theart. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al.(1989) Molecular Cloning, a laboratory manual, Cold Spring HarborLaboratories, New York, Davis et al. (1986) Basic Methods in MolecularBiology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniquescan be used to introduce one or more exogenous nucleic acids, such as anucleotide integration vector and other nucleic acid molecules, intosuitable cells (e.g., permissive cells).

A “permissive cell” refers to any cell in which a nucleic acid asdescribed by the disclosure replicates, or is capable of supportingreplication of a nucleic acid as described by the disclosure. In someembodiments, the permissive cell does not express viral capsid proteinscapable of packaging replicative copies of the nucleic acid into a viralparticle. Aspects of the disclosure relate, in part, to the surprisingdiscovery that, in some embodiments, mammalian cells are not permissivefor replication of nucleic acids described by the disclosure.Accordingly, in some embodiments, a permissive cell is a non-mammaliancell (e.g., the permissive cell is not a mammalian cell). In someembodiments, the permissive cell is an insect cell line, yeast cellline, or bacterial cell line.

Examples of permissive insect cells include but are not limited toSpodoptera frugiperda (e.g., Sf9, Sf21), Spodoptera exigua, Heliothisvirescens, Helicoverpa zea, Heliothis subflexa, Anticarsia gemmatalis,Trichopulsia ni (e.g., High-Five cells), Drosophila melanogaster (e.g.,S2, S3), Antheraea eucalypti, Bombyx mori, Aedes alpopictus, Aedesaegyptii, and others.

Examples of permissive bacterial cells include, but are not limited toEscherichia coli, Corynebacterium glutamicum, and Pseudomonasfluorescens.

Examples of permissive yeast cells include but are not limited toSaccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris, Bacillussp., Aspergillus sp., Trichoderma sp., and Myceliophthora thermophilaC1.

Examples of permissive plant cells include but are not limited toNicotiana sp., Arabidopsis thaliana, Mays zea, Solanum sp., or Lemna sp.

In some embodiments, a permissive cell is a mammalian cell. Examples ofpermissive mammalian cells include Henrietta Lacks tumor (HeLa) cellsand baby hamster kidney (BHK-21) cells.

In some embodiments, a nucleic acid as described by the disclosure iscontained within a vector and delivered to a permissive cell. As usedherein, the term “vector” includes any genetic element, such as aplasmid, phage, transposon, cosmid, chromosome, artificial chromosome,virus, virion, etc., which is capable of replication when associatedwith the proper control elements and which can transfer gene sequencesbetween cells. Thus, the term includes cloning and expression vehicles,as well as viral vectors. In some embodiments, useful vectors arecontemplated to be those vectors in which the nucleic acid segment to betranscribed is positioned under the transcriptional control of apromoter.

In some embodiments, the method comprises expressing a rolling circlereplication protein (e.g., a viral nonstructural protein, such as an AAVRep protein) in the permissive cell. In some embodiments more than one(e.g., 2, 3, 4, or more) rolling circle replication proteins areexpressed in a permissive cell. Without wishing to be bound by anyparticular theory, viral nonstructural protein(s) expressed in thepermissive cell mediate(s) replication of a nucleic acid described bythe disclosure. For example, in some embodiments, AAV Rep78 and Rep52are expressed in a permissive cell comprising a nucleic acid having AAV2ITR-based asymmetric interrupted self-complementary sequences. In someembodiments, the nonstructural viral protein is selected from the groupconsisting of AAV78, AAV52, AAV Rep68, and AAV Rep 40.

In some embodiments a rolling circle replication protein (e.g., a viralnonstructural protein, such as an AAV Rep protein) expressed in apermissive cell is encoded by a helper virus vector. As used here,“helper virus vector” refers to a viral vector that expressesmolecule(s) (e.g., one or more proteins) required for the replication ofa nucleic acid as described by the disclosure. For example, in someembodiments a helper virus vector expresses one or more rolling circlereplication proteins which bind to the RBE of an interruptedself-complementary nucleic acid sequence and initiate replication of thenucleic acid comprising the interrupted self-complementary nucleic acidsequence. Helper virus vectors are generally known and include, forexample baculovirus, adenovirus, herpesvirus, cytomegalovirus,Epstein-Barr virus, and vaccinia virus vectors. In some embodiments, ahelper virus vector is a baculovirus expression vector (BEV).Baculovirus expression vectors are generally known in the art, forexample as disclosed in Passer et al. Methods Mol Biol. 2007; 388:55-76.Examples of baculovirus vectors include but are not limited to Autographcalifornica multiple nucleopolyhedrosis virus (AcMNPV) vector, BmNPV,and Spodoptera exigua multiple nucleopolyhedrovirus. In someembodiments, the helper virus vector is Autograph californica multiplenucleopolyhedrosis virus (AcMNPV) vector.

In some embodiments, methods of producing nucleic acids described by thedisclosure further comprise a step of purifying the multiple copies ofthe nucleic acid from a cell (e.g., a permissive cell). Generally, anysuitable nucleic acid purification method can be used. For example, insome embodiments, the multiple copies of the nucleic acid described bythe disclosure are purified by plasmid purification kits, such as QiagenMiniPrep kit, ethanol precipitation, phenol-chloroform purification,etc. However, the disclosure relates, in part, to the discovery thatnucleic acids comprising interrupted self-complementary sequencesexhibit poor binding efficiency to weak cation exchange chromatographymedia (e.g., diethylaminoethyl media, DEAE) and that purification usingsilica gel media produce well-resolved molecular species while reducingthe formation of high molecular weight complexes. Thus, in someembodiments, the purification comprises contacting the nucleic acid witha silica gel resin.

In some embodiments, nucleic acids provided herein may be used todeliver a heterologous insert to a cell, e.g., for therapeutic purposes.Furthermore, in some aspects, the disclosure relates to the delivery ofnucleic acids containing heterologous inserts that encoded therapeuticproducts (e.g., therapeutic proteins, therapeutic RNAs) to a subject. Inorder to avoid administration of impure or contaminated nucleic acids orto otherwise characterize the extent or purity, quality or make up of anucleic acid preparation, such preparations may be subject to a qualitycontrol (QC) or other analysis procedure prior to use, e.g., prior toadministration to a cell or subject. For example, methods of analyzing anucleic acid, in some embodiments, comprise obtaining a nucleic acidpreparation described by the disclosure and determining a physiochemicalproperty of one or more nucleic acid components in the preparation,e.g., nucleic acid replication products.

Examples of physiochemical properties that may be determined include butare not limited to solubility, stability, structure (e.g., primarystructure, secondary structure, tertiary structure, quaternarystructure, etc.), hydrophobicity, GC content, molecular weight (e.g.,molecular weight of one or more fragments or portions of a nucleic acid,for example following restriction digest), etc. In some embodiments, thephysiochemical property is the nucleotide sequence of one or eachself-complementary sequence (e.g., determining if a nucleic aciddescribed by the disclosure comprises a truncated cross-arm sequence).

In some embodiments, the physiochemical property is the extent ofmultimerization (e.g., monomer, dimer, trimer, 4-mer, or other multimeror concatamer) of a nucleic acid as described by the disclosure. In someembodiments, the physiochemical property is the stoichiometry ofmonomeric and/or multimeric forms of the replication product in thenucleic acid preparation.

In some embodiments, the physiochemical property is the susceptibilityof one or more replication products (e.g., obtained from a nucleic acidpreparation) to digestion with a restriction endonuclease. For example,in some embodiments, a nucleic acid as described by the disclosure isdigested with one or more restriction enzymes and the fragments of thenucleic acid are analyzed to determine the size of each fragment. Thus,in some embodiments, the physiochemical property is the molecular weightof one or more replication products or of a fragment of a replicationproduct. In some embodiments, the molecular weight is of a fragment ofthe one or more replication products that comprises one or moreself-complementary sequences. In some embodiments, the molecular weightis determined based on electrophoretic mobility. In some embodiments,the molecular weight is determined based on mass spectroscopy.

In some embodiments, the molecular weight is of a fragment of the one ormore replication products. In some embodiments, the molecular weight isof fragment of a replication product that is amplified by a reactioncomprising primer extension by a polymerase. Examples ofpolymerase-based extension methods include but are not limited topolymerase chain reaction (PCR), recombinase polymerase amplification,loop mediated isothermal amplification (LAMP), etc.

In some embodiments, the physiochemical property is the polarity ofmonomers in a dimeric form of the replication product, wherein thepolarity is head-to-head, head-to-tail or tail-to-tail.

Generally, suitable assays for determining physiochemical properties maybe used. Suitable assays may include restriction digestion analysis, gelelectrophoresis (e.g., native gel electrophoresis, denaturing gelelectrophoresis, high resolution gel electrophoresis), spectrometry(e.g., mass spectrometry, such as LC/MS, HPLC/MS, ESI-MS, MALDI-TOF,etc.), and nucleic acid sequencing (e.g., Maxam-Gilbert sequencing,pyrosequencing, chain-termination sequencing, massively parallelsignature sequencing, single-molecule sequencing, nanopore sequencing,Illumina sequencing, etc.).

Compositions

In some aspects, the disclosure relates to compositions comprising anucleic acid as described by the disclosure. In some embodiments,compositions comprising nucleic acids as described herein are deliveredto a subject in need thereof. The nucleic acids may be delivered to asubject in compositions according to any appropriate methods known inthe art. It should be appreciated that compositions may comprise one ormore (e.g., a plurality) of nucleic acids as described by thedisclosure. In some embodiments, a plurality of nucleic acids is 2, 3,4, 5, 6, 7, 8, 9, 10, or more nucleic acids. In some embodiments, eachof the one or more nucleic acids of a plurality is covalently linked(e.g., linked end-to-end). In some embodiments, the composition furthercomprises a pharmaceutically acceptable carrier.

The nucleic acid, preferably suspended in a physiologically compatiblecarrier (i.e., in a composition), may be administered to a subject, i.e.host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit,horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or anon-human primate (e.g., Macaque). In some embodiments a host animaldoes not include a human.

Delivery of the nucleic acids (e.g., ceDNA) to a mammalian subject maybe by, for example, intramuscular injection or by administration intothe bloodstream of the mammalian subject. Administration into thebloodstream may be by injection into a vein, an artery, or any othervascular conduit. In some embodiments, the nucleic acids areadministered into the bloodstream by way of isolated limb perfusion, atechnique well known in the surgical arts, the method essentiallyenabling the artisan to isolate a limb from the systemic circulationprior to administration of the nucleic acid. A variant of the isolatedlimb perfusion technique, described in U.S. Pat. No. 6,177,403, can alsobe employed by the skilled artisan to administer the nucleic acid(s)into the vasculature of an isolated limb to potentially enhancetransfection of muscle cells or tissue. Moreover, in certain instances,it may be desirable to deliver the nucleic acid(s) to the CNS of asubject. By “CNS” is meant all cells and tissue of the brain and spinalcord of a vertebrate. Thus, the term includes, but is not limited to,neuronal cells, glial cells, astrocytes, cereobrospinal fluid (CSF),interstitial spaces, bone, cartilage and the like. Recombinant AAVs maybe delivered directly to the CNS or brain by injection into, e.g., theventricular region, as well as to the striatum (e.g., the caudatenucleus or putamen of the striatum), spinal cord and neuromuscularjunction, or cerebellar lobule, with a needle, catheter or relateddevice, using neurosurgical techniques known in the art, such as bystereotactic injection (see, e.g., Stein et al., J Virol 73:3424-3429,1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat.Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther.11:2315-2329, 2000).

Suitable carriers may be readily selected by one of skill in the art inview of the indication for which the nucleic acid(s) is directed. Forexample, one suitable carrier includes saline, which may be formulatedwith a variety of buffering solutions (e.g., phosphate buffered saline).Other exemplary carriers include sterile saline, lactose, sucrose,calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesameoil, and water. The selection of the carrier is not a limitation of thepresent disclosure.

Optionally, the compositions of the disclosure may contain, in additionto the nucleic acid(s) and carrier(s), other conventional pharmaceuticalingredients, such as preservatives, or chemical stabilizers. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol. Suitable chemical stabilizersinclude gelatin and albumin.

The nucleic acid(s) are administered in sufficient amounts to transfectthe cells of a desired tissue and to provide sufficient levels of genetransfer and expression without undue adverse effects. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the selected organ (e.g., intraportaldelivery to the liver), oral, inhalation (including intranasal andintratracheal delivery), intraocular, intravenous, intramuscular,subcutaneous, intradermal, intratumoral, and other parental routes ofadministration. Routes of administration may be combined, if desired.

The dose of nucleic acid(s) required to achieve a particular“therapeutic effect,” will vary based on several factors including, butnot limited to: the route of nucleic acid administration, the level ofgene or RNA expression required to achieve a therapeutic effect, thespecific disease or disorder being treated, and the stability of thegene or RNA product. One of skill in the art can readily determine anucleic acid dose range to treat a patient having a particular diseaseor disorder based on the aforementioned factors, as well as otherfactors that are well known in the art.

Dosage regime may be adjusted to provide the optimum therapeuticresponse. For example, the oligonucleotide may be repeatedlyadministered, e.g., several doses may be administered daily or the dosemay be proportionally reduced as indicated by the exigencies of thetherapeutic situation. One of ordinary skill in the art will readily beable to determine appropriate doses and schedules of administration ofthe subject oligonucleotides, whether the oligonucleotides are to beadministered to cells or to subjects.

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 70% or 80% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound in eachtherapeutically-useful composition may be prepared is such a way that asuitable dosage will be obtained in any given unit dose of the compound.Factors such as solubility, bioavailability, biological half-life, routeof administration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

In certain circumstances it will be desirable to deliver the nucleicacid-based therapeutic constructs in suitably formulated pharmaceuticalcompositions disclosed herein either subcutaneously,intraopancreatically, intranasally, parenterally, intravenously,intramuscularly, intrathecally, or orally, intraperitoneally, or byinhalation. In some embodiments, the administration modalities asdescribed in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (eachspecifically incorporated herein by reference in its entirety) may beused to deliver nucleic acids. In some embodiments, a preferred mode ofadministration is by portal vein injection.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. Dispersions may also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. In many cases the form issterile and fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art. For example, one dosage may be dissolvedin 1 ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the host. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual host.

Sterile injectable solutions are prepared by incorporating the nucleicacid in the required amount in the appropriate solvent with various ofthe other ingredients enumerated herein, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The nucleic acid compositions disclosed herein may also be formulated ina neutral or salt form. Pharmaceutically-acceptable salts, include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Supplementary active ingredients can also be incorporated into thecompositions. The phrase “pharmaceutically-acceptable” refers tomolecular entities and compositions that do not produce an allergic orsimilar untoward reaction when administered to a host.

Delivery vehicles such as liposomes, nanocapsules, microparticles,microspheres, lipid particles, vesicles, and the like, may be used forthe introduction of the compositions of the present disclosure intosuitable host cells. In particular, the nucleic acids may be formulatedfor delivery either encapsulated in a lipid particle, a liposome, avesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically acceptable formulations of the nucleic acids disclosedherein. The formation and use of liposomes is generally known to thoseof skill in the art. Recently, liposomes were developed with improvedserum stability and circulation half-times (U.S. Pat. No. 5,741,516).Further, various methods of liposome and liposome like preparations aspotential drug carriers have been described (U.S. Pat. Nos. 5,567,434;5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures. In addition,liposomes are free of the DNA length constraints that are typical ofviral-based delivery systems. Liposomes have been used effectively tointroduce genes, drugs, radiotherapeutic agents, viruses, transcriptionfactors and allosteric effectors into a variety of cultured cell linesand animals. In addition, several successful clinical trials examiningthe effectiveness of liposome-mediated drug delivery have beencompleted.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 ANG, containing an aqueous solution in the core.

In some embodiments, a liposome comprises cationic lipids. The term“cationic lipid” includes lipids and synthetic lipids having both polarand non-polar domains and which are capable of being positively chargedat or around physiological pH and which bind to polyanions, such asnucleic acids, and facilitate the delivery of nucleic acids into cells.In some embodiments, cationic lipids include saturated and unsaturatedalkyl and alicyclic ethers and esters of amines, amides, or derivativesthereof. In some embodiments, cationic lipids comprise straight-chain,branched alkyl, alkenyl groups, or any combination of the foregoing. Insome embodiments, cationic lipids contain from 1 to about 25 carbonatoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, or 25 carbon atoms. In some embodiments,cationic lipids contain more than 25 carbon atoms. In some embodiments,straight chain or branched alkyl or alkene groups have six or morecarbon atoms. A cationic lipid may also comprise, in some embodiments,one or more alicyclic groups. Non-limiting examples of alicyclic groupsinclude cholesterol and other steroid groups. In some embodiments,cationic lipids are prepared with a one or more counterions. Examples ofcounterions (anions) include but are not limited to Cl⁻, Br⁻, I⁻, F⁻,acetate, trifluoroacetate, sulfate, nitrite, and nitrate.

Non-limiting examples of cationic lipids include polyethylenimine,polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combinationof DOTMA and DOPE), Lipofectase, LIPOFECTAMINE™ (e.g., LIPOFECTAMINE™2000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), andEufectins (JBL, San Luis Obispo, Calif.). Exemplary cationic liposomescan be made from N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammoniumchloride (DOTMA), N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammoniummethylsulfate (DOTAP),3β-[N—(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol),2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; anddimethyldioctadecylammonium bromide (DDAB). Nucleic acids (e.g., ceDNA)can also be complexed with, e.g., poly (L-lysine) or avidin and lipidsmay, or may not, be included in this mixture, e.g., steryl-poly(L-lysine).

In some embodiments, a nucleic acid described by the disclosure isdelivered using a cationic lipid described in U.S. Pat. No. 8,158,601,or a polyamine compound or lipid as described in U.S. Pat. No.8,034,376, the contents of each of which are incorporated herein byreference.

In some embodiments, a nucleic acid described by the disclosure isconjugated (e.g., covalently bound to an agent that increases cellularuptake. An “agent that increases cellular uptake” is a molecule thatfacilitates transport of a nucleic acid across a lipid membrane. Forexample, a nucleic acid may be conjugated to a lipophilic compound(e.g., cholesterol, tocopherol, etc.), a cell penetrating peptide (CPP)(e.g., penetratin, TAT, Syn1B, etc.), and polyamines (e.g., spermine).Further examples of agents that increase cellular uptake are disclosed,for example, in Winkler (2013). Oligonucleotide conjugates fortherapeutic applications. Ther. Deliv. 4(7); 791-809, the contents ofwhich are incorporated herein by reference.

In some embodiments, a nucleic acid described by the disclosure isconjugated to a polymer (e.g., a polymeric molecule) or a folatemolecule (e.g., folic acid molecule). Generally, delivery of nucleicacids conjugated to polymers is known in the art, for example asdescribed in WO2000/34343 and WO2008/022309, the contents of which areincorporated herein by reference. In some embodiments, a nucleic aciddescribed by the disclosure is conjugated to a poly(amide) polymer, forexample as described by U.S. Pat. No. 8,987,377. In some embodiments, anucleic acid described by the disclosure is conjugated to a folic acidmolecule as described in U.S. Pat. No. 8,507,455, the contents of whichare incorporated herein by reference.

In some embodiments, a nucleic acid described by the disclosure isconjugated to a carbohydrate, for example as described in U.S. Pat. No.8,450,467, the contents of which are incorporated herein by reference.

Alternatively, nanocapsule formulations of the nucleic acid may be used.Nanocapsules can generally entrap substances in a stable andreproducible way. To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 μm) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use.

In some embodiments, a nucleic acid described by the disclosure isdelivered by a lipid nanoparticle. Generally, lipid nanoparticlescomprise an ionizable amino lipid (e.g.,heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate,DLin-MC3-DMA, a phosphatidylcholine(1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol and acoat lipid (polyethylene glycol-dimyristolglycerol, PEG-DMG), forexample as disclosed by Tam et al. (2013). Advances in LipidNanoparticles for siRNA delivery. Pharmaceuticals 5(3): 498-507. In someembodiments, a lipid nanoparticle has a mean diameter between about 10and about 1000 nm. In some embodiments, a lipid nanoparticle has adiameter that is less than 300 nm. In some embodiments, a lipidnanoparticle has a diameter between about 10 and about 300 nm. In someembodiments, a lipid nanoparticle has a diameter that is less than 200nm. In some embodiments, a lipid nanoparticle has a diameter betweenabout 25 and about 200 nm. In some embodiments, a lipid nanoparticlepreparation (e.g., composition comprising a plurality of lipidnanoparticles) has a size distribution in which the mean size (e.g.,diameter) is about 70 nm to about 200 nm, and more typically the meansize is about 100 nm or less.

In some embodiments, a nucleic acid described by the disclosure isdelivered by a gold nanoparticle. Generally, a nucleic acid can becovalently bound to a gold nanoparticle or non-covalently bound to agold nanoparticle (e.g., bound by a charge-charge interaction), forexample as described by Ding et al. (2014). Gold Nanoparticles forNucleic Acid Delivery. Mol. Ther. 22(6); 1075-1083. In some embodiments,gold nanoparticle-nucleic acid conjugates are produced using methodsdescribed, for example, in U.S. Pat. No. 6,812,334, the contents ofwhich are incorporated herein by reference.

In addition to the methods of delivery described above, the followingtechniques are also contemplated as alternative methods of deliveringthe nucleic acid compositions to a host. Sonophoresis (e.g., ultrasound)has been used and described in U.S. Pat. No. 5,656,016 as a device forenhancing the rate and efficacy of drug permeation into and through thecirculatory system. Other drug delivery alternatives contemplated areintraosseous injection (U.S. Pat. No. 5,779,708), microchip devices(U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al.,1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) andfeedback-controlled delivery (U.S. Pat. No. 5,697,899).

Delivery/Administration

In some embodiments, the disclosure provides a method of delivering aheterologous nucleic acid to a cell (e.g., a host cell), the methodcomprising delivering to the cell a nucleic acid as described by thedisclosure.

In some aspects, the disclosure provides transfected host cells. A “hostcell” refers to any cell that harbors, or is capable of harboring, asubstance of interest. A host cell may be used as a recipient of anucleic acid as described by the disclosure. The term includes theprogeny of the original cell which has been transfected. Thus, a “hostcell” as used herein may refer to a cell which has been transfected withan exogenous DNA sequence (e.g., a nucleic acid as described by thedisclosure). It is understood that the progeny of a single parental cellmay not necessarily be completely identical in morphology or in genomicor total DNA complement as the original parent, due to natural,accidental, or deliberate mutation.

In some embodiments, a host cell is a permissive cell. In someembodiments, a host cell is not a permissive cell. Often a host cell isa mammalian cell. In some aspects, the disclosure provides a method ofdelivering a heterologous nucleic acid to a subject comprisingadministering a host cell having a nucleic acid as described by thedisclosure to the subject. For example, in some embodiments, a host cellis a blood cell, such as a human blood cell, comprising a nucleic acidas described by the disclosure (e.g., a nucleic acid having aheterologous nucleic acid insert encoding a blood disease-associatedtransgene). Without wishing to be bound by any particular theory,delivery of such a host cell is useful, in some embodiments, fortreatment of a disease or disorder of the blood.

Aspects of the disclosure relate to the discovery that nucleic acids asdescribed herein elicit a reduced immune response (e.g., do not elicitan immune response) in a host relative to currently used viral andbacterially-derived gene therapy vectors. In some aspects, thedisclosure provides a method of delivering a heterologous nucleic acidto a subject, the method comprising delivering to the subject a nucleicacid as described by the disclosure, wherein the delivery of the nucleicacid does not result in an immune response against the nucleic acid inthe subject. In some embodiments, the immune response is a humoralresponse. Humoral immune response refers to production ofantigen-specific antibodies by B lymphocytes. In some embodiments, theimmune response is a cellular response. A cellular immune responserefers to an immune response that does not involve antibodies but ratheractivation of immune cells (e.g., phagocytes, antigen-specific T-cells,macrophages, natural killer cells, etc.) by an antigen (e.g., anexogenous nucleic acid).

Without wishing to be bound by any particular theory, the lack of immuneresponse elicited by administration of nucleic acids as described by thedisclosure allows the nucleic acids to be administered to a host onmultiple occasions. In some embodiments, the number of occasions inwhich a heterologous nucleic acid is delivered to a subject is in arange of 2 to 10 times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). Insome embodiments, a heterologous nucleic acid is delivered to a subjectmore than 10 times.

In some embodiments, a dose of nucleic acid (e.g., ceDNA) isadministered to a subject no more than once per calendar day (e.g., a24-hour period). In some embodiments, a dose of nucleic acid (e.g.,ceDNA) is administered to a subject no more than once per 2, 3, 4, 5, 6,or 7 calendar days. In some embodiments, a dose of nucleic acid (e.g.,ceDNA) is administered to a subject no more than once per calendar week(e.g., 7 calendar days). In some embodiments, a dose of nucleic acid(e.g., ceDNA) is administered to a subject no more than bi-weekly (e.g.,once in a two calendar week period). In some embodiments, a dose ofnucleic acid (e.g., ceDNA) is administered to a subject no more thanonce per calendar month (e.g., once in 30 calendar days). In someembodiments, a dose of nucleic acid (e.g., ceDNA) is administered to asubject no more than once per six calendar months. In some embodiments,a dose of nucleic acid (e.g., ceDNA) is administered to a subject nomore than once per calendar year (e.g., 365 days or 366 days in a leapyear).

As disclosed herein nucleic acids (including DNA expression constructsthat may be used to express them) may be administered by any suitableroute. For use in therapy, an effective amount of the nucleic acid(e.g., oligonucleotide) and/or other therapeutic agent can beadministered to a subject by any mode that delivers the agent to thedesired tissue, e.g., muscle tissue. In some embodiments, agents (e.g.,nucleic acids) are administered intramuscularly. Other suitable routesof administration include but are not limited to oral, parenteral,intravenous, intraperitoneal, intranasal, sublingual, intratracheal,inhalation, subcutaneous, ocular, vaginal, and rectal. Systemic routesinclude oral and parenteral. Several types of devices are regularly usedfor administration by inhalation. These types of devices include metereddose inhalers (MDI), breath-actuated MDI, dry powder inhaler (DPI),spacer/holding chambers in combination with MDI, and nebulizers.

For oral administration, the agents can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the agents of thedisclosure to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Pharmaceutical preparations fororal use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

Pharmaceutical preparations that can be used orally include push fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active agents may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. Formulations for oral administration aretypically in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, agents (e.g., nucleic acids) for useaccording to the present disclosure may be conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The agents (e.g., nucleic acids), when it is desirable to deliver themsystemically, may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of agents (e.g., antisense nucleic acids) inwater-soluble form. Additionally, suspensions of agents may be preparedas appropriate oily injection suspensions. Suitable lipophilic solventsor vehicles include fatty oils such as sesame oil, or synthetic fattyacid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances that increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents that increase the solubility of theagents to allow for the preparation of highly concentrated solutions.Alternatively, agents (e.g., nucleic acids) may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. Agents (e.g., nucleic acids) may also be formulated inrectal or vaginal compositions such as suppositories or retentionenemas, e.g., containing conventional suppository bases such as cocoabutter or other glycerides.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the agents (e.g., antisense nucleic acids),increasing convenience to the subject and the physician. Many types ofrelease delivery systems are available. They include polymer basesystems such as poly(lactide glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Delivery systems also include non-polymersystems that are: lipids including sterols such as cholesterol,cholesterol esters and fatty acids or neutral fats such as mono, di, andtri glycerides; hydrogel release systems; silastic systems;peptide-based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; andothers disclosed herein.

EXAMPLES Example 1: Interrupted Self-Complementary Nucleic AcidSequences

FIG. 1A shows a non-limiting example of an interruptedself-complementary nucleic acid sequence (which is an AAV2 ITR). Asshown in the FIG. 1A, the nucleic acid forms a T-shaped hairpinstructure having a stem portion (D-A-A′) and a cross-arm sequence havingopposing lengthwise stem-loops (B-B′ and C-C′). The trs and RBE of theinterrupted self-complementary nucleic acid sequence are also depicted.FIG. 1B shows several non-limiting examples of truncations in the“C-arm” of an interrupted self-complementary nucleic acid sequence. Agraphic depiction of one example of asymmetric AAV2 ITRs is shown inFIG. 3.

Replication of ceDNA from Asymmetric AAV ITRs

Adeno-associated virus (AAV) genomes are linear single-stranded DNAflanked by terminal palindromes typically referred to as invertedterminal repeats (ITRs) (FIG. 2A, left side). Except for 7 unpairedbases, the 145 nt ITR sequence is self-complementary and forms anenergetically stable “T” shaped hairpin (ΔG≈−72.4 kcal per mol, Tm>80°C.) (FIG. 1A). During virus DNA replication, the cellular DNA polymerasecomplex initiates synthesis at the 3′-terminus of the template strandwhere the partial duplex formed by the ITR serves as the primer forprimer extension. The replicative intermediate formed is anintramolecular duplex with the template and nascent strands covalentlyconnected by the ITR. During a productive infection, a process calledterminal resolution resolves the intra-strand ITR resulting in twofull-length, complementary virus genomes (FIG. 2B, left side).

In the absence of AAV cap gene expression, an AAV vector genome havingasymmetrical ITRs undergoes inefficient replication, and the replicationproducts accumulate in a novel conformation of closed-ended linearduplex DNA (ceDNA). Due to inefficient replication (e.g., incompleteterminal resolution), the complementary strands of the intramolecularintermediate are now covalently linked through the ITRs on both ends ofthe genome (FIG. 2B, right side). Thus, in native conditions, ceDNAbehaves as linear duplex DNA, however, in denaturing conditions, ceDNAstrands melt apart, but remain linked, therefore transforming the linearduplex molecule into single-stranded circular DNA.

In some embodiments, ceDNA production from asymmetric AAV ITRs isdependent on a truncated ITR at one end and an operative (e.g.,functional), wild-type (wt) or wt-like ITR on the opposite end of thetransgene cassette. In some embodiments, the truncated ITR isinefficiently processed during the replication cycles of ceDNA in theSf9 cells leading to an accumulation of replication intermediates,duplex monomer, duplex dimers, etc. In the absence of structural(capsid) protein expression, Rep 78 (or Rep 68) assembles on the intactITRs and catalyze the site-specific nicking at the terminal resolutionsite. The reaction results in the formation of a transienttyrosine-phosphodiester (Y156 for AAV2 Rep 78 covalently linked to the5′ thymidine of the terminal resolution site 5′ΔGTTGG). The transientnucleoprotein complex then transfers the donor strand to the free3′-terminus of the complementary strand. The ceDNA conformationtherefore, results from the defective ITR on one end of the vectorgenome, an intact or operative ITR on the opposite end, and theco-expression of the p5 and p19 Rep proteins, where at least one p5 andone p19 Rep are expressed. Other parvovirus “small” Rep or NS proteinscan substitute for AAV p19 Rep protein (Rep 52 and Rep 40). Since thesesmall Rep proteins are non-processive, monomeric helicases, it isfeasible that non-parvoviridae super family 2 (SF2) helicases cansubstitute for the AAV Rep proteins.

The dependovirinae have existed relatively unchanged for tens ofmillions (and likely, hundreds of millions) of years. It is hypothesizedthat they have developed a homeostatic or symbiotic relationship withtheir hosts in which the provirus in latently infected cells oftenlocalizes to the AAVS1 locus on human chromosome 19q, which appears tohave no adverse effect on the host cell. While in latency, AAV geneexpression is repressed by cellular factors, e.g., YY1, and by the p5Rep proteins that acting servomechanistically, negatively regulateexpression from the p5 and p40 promoters. Thus, latently infected cellshave little or no detectable AAV proteins and limit the acquired immuneresponse to cells that are synthesizing virus proteins.

Example 2: Purification of ceDNA

Plasmid purification protocols typically utilize silica gels for smallplasmid quantities preparations or anion exchange chromatographydiethylaminoethyl sepharo se, (DEAE-sepharose, for example) for largerquantities. For large-scale pDNA purification, the Escherichia colibacteria are lysed with sodium dodecyl sulfate (SDS) and the cellproteins and genomic DNA are precipitated by denaturation/neutralizationcycle retaining the plasmid in solution. The renatured double-strandedpDNA binds to the positively charged DEAE group and following washingsteps, the pDNA is displaced and eluted with a high salt buffersolution. Alternatively, silica gel membranes may be used for selectivepDNA adsorption from the clarified bacterial lysate under high saltconditions and eluted in low or no iconicity buffer.

It was observed that ceDNA can be readily purified with the small scalesilica gel-based process. Using the large-scale anion exchange protocol,ceDNA recovery was very inefficient and resulted in low yields.

A method for large-scale ceDNA purification from invertebrate Sf9 cellsusing silica gel membranes is described. Increasing the surface area ofmodified silica gel membrane (or chromatography medium) is one approachto improve the flow rate, adsorption, and recovery of ceDNA. Standardfilter capsules are available in a wide-range of configurations rangingin size from 0.5 cm diameter to 20 cm diameter or more. The capsule maycontain a single layer of silica gel membrane on a chemically inertsupport, or stacks of membranes separated by inert supports, or pleatedmembranes utilizing horizontal flow design. Tangential flow filtration(TFF) and hollow fiber filtration (HFF) are alternatives for the coaxialflow capsule filtration.

Example 3: ceDNA Constructs for Treatment of Disease

FIG. 4 shows non-limiting examples of nucleic acid constructs havingasymmetric self-complementary nucleic acid sequences (e.g., AAV2 ITRs)and a transgene associated with ocular disease. The first nucleic acidconstruct of FIG. 4 depicts a non-limiting example of a nucleic acidmolecule for treatment of Stargardt's disease. The nucleic acidcomprises a pair of asymmetrical interrupted self-complementary nucleicacid sequences (e.g., asymmetric AAV2 ITRs) flanking a heterologousnucleic acid insert encoding the ATP-Binding Cassette, Sub-Family A(ABC1), Member 4 (ABCA4) protein. The AAV2 ITRs are asymmetric becauseone of the AAV2 ITRs (right side) contains a deletion in the C-stemregion. Each interrupted self-complementary nucleic acid sequenceincludes an operative Rep-binding element (RBE) and an operativeterminal resolution site (trs). The nucleic acid also encodes a hBglobinintron 2 positioned 5′ to the heterologous nucleic acid insert, and ahGH poly(A) signal positioned 3′ to the heterologous nucleic acidinsert.

The second nucleic acid construct in FIG. 4 depicts a non-limitingexample of a nucleic acid molecule for treatment of Usher's disease. Thenucleic acid comprises a pair of asymmetrical interruptedself-complementary nucleic acid sequences (e.g., asymmetric AAV2 ITRs)flanking a heterologous nucleic acid insert encoding usherin protein(USH2A) variant 1. The AAV2 ITRs are asymmetric because one of the AAV2ITRs (right side) contains a deletion in the C-stem region. Eachinterrupted self-complementary nucleic acid sequence includes anoperative Rep-binding element (RBE) and an operative terminal resolutionsite (trs). The nucleic acid also encodes a CMV promoter and a T7promoter positioned 5′ to the heterologous nucleic acid insert, and abGH poly(A) signal positioned 3′ to the heterologous nucleic acidinsert.

The third nucleic acid construct in FIG. 4 depicts a non-limitingexample of a nucleic acid molecule for treatment of maculardegeneration/neovascularization. The nucleic acid comprises a pair ofasymmetrical interrupted self-complementary nucleic acid sequences(e.g., asymmetric AAV2 ITRs) flanking a heterologous nucleic acid insertencoding vascular endothelial growth factor receptor (VEGFR). The AAV2ITRs are asymmetric because one of the AAV2 ITRs (right side) contains adeletion in the C-stem region. Each interrupted self-complementarynucleic acid sequence includes an operative Rep-binding element (RBE)and an operative terminal resolution site (trs). The nucleic acid alsoencodes a CMV promoter and a T7 promoter positioned 5′ to theheterologous nucleic acid insert, and a bGH poly(A) signal positioned 3′to the heterologous nucleic acid insert.

The fourth nucleic acid construct in FIG. 4 depicts a non-limitingexample of a nucleic acid molecule for treatment of Leber's congenitalamaurosis. The nucleic acid comprises a pair of asymmetrical interruptedself-complementary nucleic acid sequences (e.g., asymmetric AAV2 ITRs)flanking a heterologous nucleic acid insert encoding centrosomal protein290 (CEP290). The AAV2 ITRs are asymmetric because one of the AAV2 ITRs(right side) contains a deletion in the C-stem region. Each interruptedself-complementary nucleic acid sequence includes an operativeRep-binding element (RBE) and an operative terminal resolution site(trs). The nucleic acid also encodes a CMV promoter and a T7 promoterpositioned 5′ to the heterologous nucleic acid insert, and a bGH poly(A)signal positioned 3′ to the heterologous nucleic acid insert.

FIG. 5 shows non-limiting examples of nucleic acid constructs havingasymmetric self-complementary nucleic acid sequences (e.g., AAV2 ITRs)and a transgene associated with blood disease. The first nucleic acidconstruct in FIG. 5 depicts a non-limiting example of a nucleic acidmolecule for treatment of Hemophilia A. The nucleic acid comprises apair of asymmetrical interrupted self-complementary nucleic acidsequences (e.g., asymmetric AAV2 ITRs) flanking a heterologous nucleicacid insert encoding B-domain deleted factor VIII protein (BDD-FVIII).The AAV2 ITRs are asymmetric because one of the AAV2 ITRs (right side)contains a deletion in the C-stem region. Each interruptedself-complementary nucleic acid sequence includes an operativeRep-binding element (RBE) and an operative terminal resolution site(trs). The nucleic acid also encodes a CMV promoter/enhancer and a T7promoter positioned 5′ to the heterologous nucleic acid insert, and abGH poly(A) signal positioned 3′ to the heterologous nucleic acidinsert.

The second nucleic acid construct in FIG. 5 depicts a non-limitingexample of a nucleic acid molecule for treatment of Hemophilia A. Thenucleic acid comprises a pair of asymmetrical interruptedself-complementary nucleic acid sequences (e.g., asymmetric AAV2 ITRs)flanking a heterologous nucleic acid insert encoding full-length factorVIII protein (FVIII), which exceeds the cloning capacity of traditionalAAV vectors. The AAV2 ITRs are asymmetric because one of the AAV2 ITRs(right side) contains a deletion in the C-stem region. Each interruptedself-complementary nucleic acid sequence includes an operativeRep-binding element (RBE) and an operative terminal resolution site(trs). The nucleic acid also encodes a CMV promoter/enhancer and a T7promoter positioned 5′ to the heterologous nucleic acid insert, and abGH poly(A) signal positioned 3′ to the heterologous nucleic acidinsert.

The third nucleic acid construct in FIG. 5 depicts a non-limitingexample of a nucleic acid molecule for treatment of von Willebrandfactor disease. The nucleic acid comprises a pair of asymmetricalinterrupted self-complementary nucleic acid sequences (e.g., asymmetricAAV2 ITRs) flanking a heterologous nucleic acid insert encoding vonWillebrand factor (vWF). The AAV2 ITRs are asymmetric because one of theAAV2 ITRs (right side) contains a deletion in the C-stem region. Eachinterrupted self-complementary nucleic acid sequence includes anoperative Rep-binding element (RBE) and an operative terminal resolutionsite (trs). The nucleic acid also encodes a CMV promoter and a T7promoter positioned 5′ to the heterologous nucleic acid insert, and abGH poly(A) signal positioned 3′ to the heterologous nucleic acidinsert.

FIG. 6 shows non-limiting examples of nucleic acid constructs havingasymmetric self-complementary nucleic acid sequences (e.g., AAV2 ITRs)and a transgene associated with liver disease. The first nucleic acidconstruct in FIG. 6 depicts a non-limiting example of a nucleic acidmolecule for treatment of hypercholesterolemia. The nucleic acidcomprises a pair of asymmetrical interrupted self-complementary nucleicacid sequences (e.g., asymmetric AAV2 ITRs) flanking a heterologousnucleic acid insert encoding lecithin cholesterol acetyl transferase.The AAV2 ITRs are asymmetric because one of the AAV2 ITRs (right side)contains a deletion in the C-stem region. Each interruptedself-complementary nucleic acid sequence includes an operativeRep-binding element (RBE) and an operative terminal resolution site(trs). The nucleic acid also encodes a CMV promoter/enhancer and a T7promoter positioned 5′ to the heterologous nucleic acid insert, and abGH poly(A) signal positioned 3′ to the heterologous nucleic acidinsert.

The second nucleic acid construct in FIG. 6 depicts a non-limitingexample of a nucleic acid molecule for treatment of phenylketonuria(PKU). The nucleic acid comprises a pair of asymmetrical interruptedself-complementary nucleic acid sequences (e.g., asymmetric AAV2 ITRs)flanking a heterologous nucleic acid insert encoding phenylalaninehydroxylase (PAH). The AAV2 ITRs are asymmetric because one of the AAV2ITRs (right side) contains a deletion in the C-stem region. Eachinterrupted self-complementary nucleic acid sequence includes anoperative Rep-binding element (RBE) and an operative terminal resolutionsite (trs). The nucleic acid also encodes a CMV promoter/enhancer and aT7 promoter positioned 5′ to the heterologous nucleic acid insert, and abGH poly(A) signal positioned 3′ to the heterologous nucleic acidinsert.

The third nucleic acid construct in FIG. 6 depicts a non-limitingexample of a nucleic acid molecule for treatment of Glycogen StorageDisease 1 (GSD1). The nucleic acid comprises a pair of asymmetricalinterrupted self-complementary nucleic acid sequences (e.g., asymmetricAAV2 ITRs) flanking a heterologous nucleic acid insert encoding glucose6-phosphatase catalytic subunit (G6PC). The AAV2 ITRs are asymmetricbecause one of the AAV2 ITRs (right side) contains a deletion in theC-stem region. Each interrupted self-complementary nucleic acid sequenceincludes an operative Rep-binding element (RBE) and an operativeterminal resolution site (trs). The nucleic acid also encodes a CMVpromoter/enhancer and a T7 promoter positioned 5′ to the heterologousnucleic acid insert, and a bGH poly(A) signal positioned 3′ to theheterologous nucleic acid insert.

FIG. 7 shows non-limiting examples of nucleic acid constructs havingasymmetric self-complementary nucleic acid sequences (e.g., AAV2 ITRs)and a transgene associated with lung disease. The first nucleic acidconstruct in FIG. 7 depicts a non-limiting example of a nucleic acidmolecule for treatment of cystic fibrosis. The nucleic acid comprises apair of asymmetrical interrupted self-complementary nucleic acidsequences (e.g., asymmetric AAV2 ITRs) flanking a heterologous nucleicacid insert encoding cystic fibrosis transmembrane conductance regulator(CFTR). The AAV2 ITRs are asymmetric because one of the AAV2 ITRs (rightside) contains a deletion in the C-stem region. Each interruptedself-complementary nucleic acid sequence includes an operativeRep-binding element (RBE) and an operative terminal resolution site(trs). The nucleic acid also encodes a CMV promoter/enhancer and a T7promoter positioned 5′ to the heterologous nucleic acid insert, and abGH poly(A) signal positioned 3′ to the heterologous nucleic acidinsert.

The second nucleic acid construct in FIG. 7 depicts a non-limitingexample of a nucleic acid molecule for treatment of alpha-1 antitrypsindeficiency. The nucleic acid comprises a pair of asymmetricalinterrupted self-complementary nucleic acid sequences (e.g., asymmetricAAV2 ITRs) flanking a heterologous nucleic acid insert encoding alpha-1antitrypsin (AAT). The AAV2 ITRs are asymmetric because one of the AAV2ITRs (right side) contains a deletion in the C-stem region. Eachinterrupted self-complementary nucleic acid sequence includes anoperative Rep-binding element (RBE) and an operative terminal resolutionsite (trs). The nucleic acid also encodes a CMV promoter/enhancer and aT7 promoter positioned 5′ to the heterologous nucleic acid insert, and abGH poly(A) signal positioned 3′ to the heterologous nucleic acidinsert.

Example 4: In Vivo Studies: Hydrodynamic Delivery of Naked DNA in NormalMale ICR Mice

After infusing pDNA into mouse tail veins, lacZ expression decreasedcontinuously between 24 hr and 5 weeks, corresponding to the quantity ofpDNA per diploid genome. Mouse livers treated with ceDNA havingasymmetric interrupted self-complementary sequences showed no change inlacZ activity between 24 hr and 1 week, however expression wassubstantially reduced at 5 wks. However, ceDNA in hepatocytes remainedessentially unchanged at the 5 wk point. Similar results were obtainedwith thyroxine binding globulin promoter (TBG) GFP cassettes: little orno GFP expression and pDNA were detectable after 10 wks, whereas ceDNATBG-GFP expression and DNA levels remained unchanged between 1 and 10weeks.

Example 5: Ocular Delivery of ceDNA

Direct GFP fluorescence was observed uniformly in cells spanning thewidth of the retina. FIGS. 8A-8D show delivery of ceDNA havingasymmetric interrupted self-complementary sequences to the eye. Adultmice were anesthetized by Ketamine/Xylazine (100/10 mg/kg) and thetransfection agent was delivered intravitreally by a trans scleralinjection of a volume of 1-2 μl. Antibiotic ointment was applied oncornea to prevent eye from drying while the mouse was recovering. Mousewas allowed to recover at 37 degree and then placed back into the mouseroom for 2 weeks and the euthanized by CO₂ asphyxiation. Retina wasdissected and processed for flat mount or section. No GFP antibodystaining was necessary to detect transfected cells. FIG. 8A shows a flatmount of GFP fluorescence on a mouse retina. FIG. 8B shows GFPfluorescence in a cross section of the retina. FIG. 8C shows GFPfluorescence and glial cell staining in a cross section of the retina.FIG. 8D shows GFP fluorescence in mouse retina after delivery of ceDNAby sub-retinal electroporation (top) and intravitreal injection(bottom).

Example 6. In Vivo Studies: Intrastriatal Delivery in Rats

ceDNA-GFP (ceDNA having asymmetric interrupted self-complementarysequences) was formulated with a commercially available transfectionreagent, in vivo jetPEI, under an appropriate concentration (PolyplusCorp.) and injected into intracranially into rat striatum (FIG. 9A).Rats were sacrificed 3 wks and 20 wks post-injection and followingprocessing, brain sections were examined using immunohistochemistry(IHC) with antibodies (Abs) against GFP, MHCII, and Iba1. Similar GFPexpression was seen at 3 wks and 20 wks. At 3 wks, no MHCII or Iba1antigen was detected in the brain sections, as shown in FIGS. 9B-9C.

Example 7: Sf9 Infection to Produce ceDNA

Transposition into Bacmid

DH10Bac competent cells (Invitrogen) were thawed on ice. 50 μl of cellswere dispensed into 14 ml tubes, add 50 ng of plasmid DNA (e.g., aplasmid comprising a protein-encoding transgene flanked by asymmetricinterrupted self-complementary sequences) and gently mix. The DNAsequence of the plasmid described in this example is represented by SEQID NO: 25. The DNA sequence of the region of the plasmid comprising aprotein-encoding transgene flanked by asymmetric interruptedself-complementary sequences is represented by SEQ ID NO: 26

Sequence analysis of the plasmid DNA interrupted self-complementarysequences is shown in FIGS. 10A-10B. The 5′ self-complementary sequence(referred to as the head portion, which is upstream of the codingsequence of the transgene) is shown in FIG. 10A. This was sequencedusing a primer complementary with the CMV promoter of the plasmidconstruct. FIG. 10A shows a restriction map of the plasmid in the regionof the 5′ self-complementary sequence. Two runs of the plasmid sequenceresults are aligned with a reference sequence, which is a wild-type AAV2ITR sequence.

The 3′ self-complementary sequence (referred to as the tail portion,which is downstream of the coding sequence of the transgene) is shown inFIG. 10B. This was sequenced using a primer complementary with the SV40promoter of the plasmid construct. FIG. 10B shows a restriction map ofthe plasmid in the region of the 3′ self-complementary sequence. Tworuns of the plasmid sequence results are aligned with a referencesequence, which is a wild-type AAV2 ITR sequence. The results show atruncation within the self-complementary sequence with corresponds toone arm of the cross-arm structure.

These results confirm that the plasmid DNA construct encodes a transgeneflanked by asymmetric interrupted self-complementary sequences. Theasymmetry arises because the 5′ self-complementary sequence encodes acomplete cross-arm; whereas the 3′ self-complementary sequence encodes atruncated cross-arm.

The tubes were then incubated on ice for 30 min and then heat shocked in42° C. water bath for 45 sec. Tubes were then chilled on ice for 2 min.450 μl of SOC media was added to the tubes and they were shaken in a 37°C. incubator for 4 hrs. Aliquots were diluted 1:10 and 50 μl were platedon agar plates containing 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10μg/ml tetracycline, 100 μg/ml Bluo-gal and 40 μg/ml IPTG. Plates wereincubated 48 hrs at 37° C. or 72 hrs at 30° C. A single white colony waspicked, and struck on a fresh plate to make sure it was not contaminatedwith a blue colony, then incubated overnight. Glycerol stocks werecreated.

Prepare DNA from Bacmid

The bacmid was incubated into 10 ml LB media with 50 μg/ml kanamycin, 7μg/ml gentamycin, 10 μg/ml tetracycline and grown in a 37° C. shakingincubator overnight. The culture was spun down at 8000 rpm for 10 min.The supernatant was removed and the pellet was resuspended in 1.5 ml ofP1 buffer from a Qiagen extraction kit. 1.5 ml of Buffer 2 was added andthe tube was inverted 4-6 times. 2.1 ml of Buffer P3 was added and thetube was inverted 4-6 times. The precipitants were passed through aQiagen syringe filter and centrifuged 8000 rpm for 10 min. The cleanlysis solution was transferred to a tube containing 5 ml of isopropanoland mixed well and centrifuged at 4° C. for 30 min at maximum speed. Thesupernatant was removed and 3 ml of 70% ethanol was added to wash thepellet. The pellet was then spun at 8000 rpm for 10 min. The pellet wasthen washed and air dried. The DNA was then resuspended in 200 μl TEbuffer. An aliquot was removed and optical density (OD) at 260 nm wasread to quantify DNA concentration.

Production of P1 Baculovirus Expression Vectors (BEV)

A total of 4×10⁶ Sf9 cells were seeded in 5 ml of media in T25 TC flask.The flask was placed on a horizontal surface during cell attachment sothat the cells will be uniformly distributed. Generally, cells attachwithin 15 min. 1 μg of Bacmid DNA was diluted in 75 μl of water. 6 μl ofPromega Fugene HD was diluted into 69 μl of water in a separate tube.The diluted Fugene HD was mixed with diluted DNA by pipetting up anddown several times. After 15 min, the DNA/lipid complexes were added tothe Sf9 cells. P1 baculovirus expression vector (BEV) were harvestedafter 4 days. Briefly, all media was removed from the flask using a 5 mlpipette and transferred to 15 ml a polystyrene tube. The container wasspun at 1200 g for 10 min to pellet any cells or debris that might havebeen picked up. BEV were decanted into a fresh 15 ml tube and virus wasstored at 4° C.

Production of Baculovirus-Infected Insect Cells (Biics) 50 ml of Sf9cells were infected at a concentration of 2.5×10⁶ cells/ml with 1 ml ofBEV (1:50). Cells were counted and their diameter was checked on day 1(e.g., checked to see if the cell count reaches 2×10⁶/ml and cellsmeasure ˜15-16 μm in diameter) and day 2 (e.g., checked to see if thecell count reaches between 4-5×10⁶ cells/ml and the diameter measures˜18-19 μm). Cells were spun down when they look infected at 300×g for 5min. The supernatant was removed and the pellet was resuspended infreezing media to have a final concentration of 20×10⁷ cells/ml. Cellscan be frozen at −80° C. in rack with isopropanol for storage.

Test Efficiency of Infectivity for Titerless Infected Baculovirus Stock(TIPS)

Four flasks of 20 ml at 2.5×10⁶ cells/ml were prepared. Cells wereinfected with 4 different dilutions of titerless infected baculovirusstock (TIPS), for example 1/1,000, 1/10,000, 1/50,000, 1/100,000. Cellswere grown and diameter, viability and cell counts were assessed for 3days. To determine the efficiency of TIPS stock check which dilutionreached the following criteria after 3 days: viable cells 4 to 5×10⁵cells/ml, viability 85 to 95%, diameter 18 to 20 μm.

Production of ceDNA:

2.5×10⁶ cells/ml Sf9 cells were co-infected with TIPS for Rep proteinand transgene of interest (e.g., GFP). After 4-5 days cells wereobserved for diameter and viability. Cells in the pellet were collectedby spinning down in a centrifuge at 4150 rpm for 30 min. Cells were thenfrozen or ceDNA was extracted, for example by Qiagen Midi Pluspurification protocol.

Analysis of ceDNA

ceDNA were analyzed by native gel electrophoresis and/or denaturing gelelectrophoresis, with restriction digestion. For example a single cutrestriction enzyme approximately ⅔ into the ceDNA sequence between ITRswas selected, and the resulting restriction digest products were run onan agarose gel.

FIG. 10C shows the ˜4.5 kb of a ceDNA comprising a GFP transgene(ceDNA-GFP) was electrophoretically separated into different conformers(e.g., monomer, dimer, trimer, etc.) under native (left) and denaturing(right) conditions, following digestion with the single-cutter, XhoI.

On a native gel (FIG. 10C, left), the monomers were resolved into twoproducts: 2.1 kb and 0.4 kb. The dimers were resolved into either 4.1kb/0.4 kb products, or 4.1 kb/0.8 kb products, depending upon theorientation (e.g., tail-to-tail, or head-to-head) of the dimer subunits.The products that result from tail-to-tail organization of the dimerwere indicated by the downward pointing arrows. The 0.4 kb terminalfragment was obscured by the fluorescence of the impurities at thebottom of the gel. The upward facing arrows indicate the productsresulting from the head-to-head dimer. Heavy weighted lines indicate theposition of the truncated ITR. In the monomer, the deleted ITR was onthe right side of the ceDNA molecule and in the tail-to-tail dimer, thetruncated ITR was internal (at the mirror plane). In the head-to-headconformation, the truncated ITR would be at the ends of the molecule.

On a denaturing gel (FIG. 10C, right), the dimeric ceDNA-GFP produced aband at 4.1 kb and 0.8 kb (which correlates to the denatured 0.4 kbterminal products running on the gel as a 0.8 kb single-stranded DNA).As described above, the predicted products for head-to-head ceDNA-GFPdimers were 0.8 kb and 4.1 kb on native gels, and 0.8 kb and 8.2 kb ondenaturing gels. The denaturing gel shows no band at 8.2 kb andtherefore indicates that the predominant form of the ceDNA-GFP dimer wastail-to-tail.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,and/or methods, if such features, systems, articles, materials, and/ormethods are not mutually inconsistent, is included within the scope ofthe present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

What is claimed is:
 1. A nucleic acid comprising a heterologous nucleicacid insert flanked by at least one interrupted self-complementarysequence, each self-complementary sequence having an operative terminalresolution site and a rolling circle replication protein bindingelement, wherein the self-complementary sequence is interrupted by across-arm sequence forming two opposing, lengthwise-symmetricstem-loops, each of the opposing lengthwise-symmetric stem-loops havinga stem portion in the range of 5 to 15 base pairs in length and a loopportion having 2 to 5 unpaired deoxyribonucleotides.
 2. The method ofclaim 1, wherein a second interrupted self-complementary sequence isinterrupted by a truncated cross-arm sequence.
 3. A nucleic acidcomprising a heterologous nucleic acid insert flanked by interruptedself-complementary sequences, each self-complementary sequence having anoperative terminal resolution site and a rolling circle replicationprotein binding element, wherein one self-complementary sequence isinterrupted by a cross-arm sequence forming two opposing,lengthwise-symmetric stem-loops, each of the opposinglengthwise-symmetric stem-loops having a stem portion in the range of 5to 15 base pairs in length and a loop portion having 2 to 5 unpaireddeoxyribonucleotides, wherein the other of the self-complementarysequences is interrupted by a truncated cross-arm sequence.
 4. Thenucleic acid of claim any of claims 1 to 3, wherein the interruptedself-complementary sequence(s) are in the range of 40 to 1000nucleotides in length.
 5. The nucleic acid of claim 4, wherein theinterrupted self-complementary sequence(s) are in the range of 100 to160 nucleotides in length.
 6. The nucleic acid of any one of claims 1 to5, wherein the cross-arm sequence has a Gibbs free energy (ΔG) ofunfolding under physiological conditions in the range of −12 kcal/mol to−30 kcal/mol.
 7. The nucleic acid of claim 6, wherein the cross-armsequence has a Gibbs free energy (ΔG) of unfolding under physiologicalconditions in the range of −20 kcal/mol to −25 kcal/mol.
 8. The nucleicacid of any preceding claim, wherein each of the opposinglengthwise-symmetric stem-loops have a stem portion in the range of 3 to15 base pairs in length.
 9. The nucleic acid of any preceding claim,wherein each of the opposing lengthwise-symmetric stem-loops have a stemportion in the range of 8 to 10 base pairs in length.
 10. The nucleicacid of any preceding claim, wherein each loop portion has 2 to 5unpaired deoxyribonucleotides.
 11. The nucleic acid of any precedingclaim, wherein each loop portion has three deoxyribonucleotides.
 12. Thenucleic acid of any preceding claim, wherein one loop portion has threedeoxythymidines and the other loop portion has three deoxyadenosines.13. The nucleic acid of any preceding claim, wherein the rolling circlereplication protein binding element is a Rep binding element (RBE). 14.The nucleic acid of claim 13, wherein the RBE comprises the sequence5′-GCTCGCTCGCTC-3′.
 15. The nucleic acid of any preceding claim, whereinthe operative terminal resolution site comprises a sequence 5′-TT-3′.16. The nucleic acid of any preceding claim, wherein the 3′ end of theoperative terminal resolution site is 15 to 25 nucleotides from the 5′end of the rolling circle replication protein binding element.
 17. Thenucleic acid of any one of claims 2-16, wherein the truncated cross-armsequence forms two opposing, lengthwise-asymmetric stem-loops.
 18. Thenucleic acid of claim 17, wherein one of the opposing,lengthwise-asymmetric stem-loops has a stem portion in the range of 8 to10 base pairs in length and a loop portion having 2 to 5 unpaireddeoxyribonucleotides.
 19. The nucleic acid of claim 18, wherein the onelengthwise-asymmetric stem-loop has a stem portion less than 8 basepairs in length and a loop portion having 2 to 5 deoxyribonucleotides.20. The nucleic acid of claim 19, wherein the one lengthwise-asymmetricstem-loop has a stem portion less than 3 base pairs in length.
 21. Thenucleic acid of claim 18, wherein the one lengthwise-asymmetricstem-loops has a loop portion having 3 or fewer deoxyribonucleotides.22. The nucleic acid of any one of claims 17 to 21, wherein thetruncated cross-arm sequence has a Gibbs free energy (ΔG) of unfoldingunder physiological conditions in the range of 0 kcal/mol to greaterthan −22 kcal/mol.
 23. The nucleic acid of any preceding claim, whereinthe heterologous nucleic acid insert is engineered to express a proteinor functional RNA.
 24. The nucleic acid of any preceding claim, whereinthe heterologous nucleic acid insert is a promoterless construct as asubstrate for gene editing.
 25. The nucleic acid of claim 24, whereinthe promoterless construct provides a substrate for TALENS, zinc fingernucleases (ZFNs), meganucleases, Cas9, and other gene editing proteins.26. The nucleic acid of any preceding claim, wherein the nucleic acid isin the range of 500 to 50,000 nucleotides in length.
 27. The nucleicacid of any preceding claim, wherein the nucleic acid comprises a singleclosed-ended strand.
 28. A composition comprising a plurality of nucleicacids as described in any one of claims 1 to
 27. 29. The composition ofclaim 29, wherein the plurality of nucleic acids are linked end-to-end.30. A composition comprising a nucleic acid as described in any one ofclaims 1 to 27 and a pharmaceutically acceptable carrier.
 31. A hostcell comprising the nucleic acid of any one of claims 1 to
 27. 32. Thehost cell of claim 31, wherein the host cell further comprises a rollingcircle replication protein that selectively binds to the rolling circlereplication protein binding element of the nucleic acid.
 33. A method ofdelivering a heterologous nucleic acid to a cell, the method comprisingdelivering to the cell a nucleic acid of any one of claims 1 to
 27. 34.A method of delivering a heterologous nucleic acid to a subject, themethod comprising delivering to the subject a nucleic acid of any one ofclaims 1 to 27, wherein the delivery of the nucleic acid does not resultin an immune response against the nucleic acid in the subject.
 35. Themethod of claim 34, wherein the immune response is a humoral response.36. The method of claim 34, wherein the immune response is a cellularresponse.
 37. The method of claim 34, wherein the heterologous nucleicacid is delivered on multiple occasions to the subject.
 38. The methodof claim 34, wherein the number of occasions in which heterologousnucleic acid is delivered to the subject is in a range of 2 to 10 times.39. The method of claim 37 or 38, wherein the heterologous nucleic acidis delivered to the subject hourly, daily, weekly, biweekly, monthly,quarterly, semi-annually, or annually.
 40. A method of delivering aheterologous nucleic acid to a subject, the method comprising deliveringa host cell of claim 31 or 32 to the subject.
 41. The method of claim40, wherein the host cell is a blood cell.
 42. The method of claim 40 or41, wherein the host cell is delivered on multiple occasions.
 43. Themethod of claim 42, wherein the host cell is delivered to the subjecthourly, daily, weekly, biweekly, monthly, quarterly, semi-annually, orannually.
 44. A method of preparing nucleic acids, the methodcomprising: (i) introducing into a permissive cell a nucleic acidencoding a heterologous nucleic acid insert flanked by at least oneinterrupted self-complementary sequence, each self-complementarysequence having an operative terminal resolution site and a rollingcircle replication protein binding element, wherein theself-complementary sequence is interrupted by a cross-arm sequenceforming two opposing, lengthwise-symmetric stem-loops, each of theopposing lengthwise-symmetric stem-loops having a stem portion in therange of 5 to 15 base pairs in length and a loop portion having 2 to 5unpaired deoxyribonucleotides; and (ii) maintaining the permissive cellunder conditions in which a rolling circle replication protein in thepermissive cell initiates production of multiple copies of the nucleicacid, wherein the permissive cell does not express viral capsid proteinscapable of packaging replicative copies of the nucleic acid into a viralparticle.
 45. The method of claim 44, further comprising the step ofpurifying the multiple copies of the nucleic acid.
 46. The method ofclaim 44, wherein the step of purifying comprises contacting the nucleicacid with a silica gel resin.
 47. The method of any one of claims 44 to46, wherein the rolling circle replication protein is selected from thegroup consisting of AAV78, AAV52, AAV Rep68, and AAV Rep
 40. 48. Themethod of any one of claims 44 to 47, wherein the permissive cell is nota mammalian cell.
 49. The method of claim 48, wherein the permissivecell is an insect cell line, yeast cell line, or bacterial cell line.50. The method of any one of claims 44-49, wherein the rolling circlereplication protein is encoded by a helper virus vector, optionallywherein the helper virus vector is Autograph californica multiplenucleopolyhedrosis virus (AcMNPV) vector or a baculovirus expressionvectors (BEV).
 51. A composition comprising: i) a monomeric nucleic acidcomprising a single subunit and ii) at least one multimeric nucleic acidcomprising two or more subunits, wherein each subunit of the monomericnucleic acid and of the at least one multimeric nucleic acid comprises aheterologous nucleic acid insert flanked by interruptedself-complementary sequences, each self-complementary sequence having anoperative terminal resolution site and a rolling circle replicationprotein binding element, wherein one self-complementary sequence isinterrupted by a cross-arm sequence forming two opposing,lengthwise-symmetric stem-loops and the other of the self-complementarysequences is interrupted by a truncated cross-arm sequence.
 52. Thecomposition of claim 51, wherein the at least one multimeric nucleicacid is a comprises two subunits.
 53. The composition of claim 52,wherein the two subunits are linked in a tail-to-tail configuration. 54.A method of preparing nucleic acids, the method comprising: (i)introducing into a permissive cell a nucleic acid comprising aheterologous nucleic acid insert flanked by interruptedself-complementary sequences, each self-complementary sequence having anoperative terminal resolution site and a rolling circle replicationprotein binding element, wherein one self-complementary sequence hasbeen determined to be interrupted by a cross-arm sequence that forms twoopposing, lengthwise-symmetric stem-loops, wherein the other of theself-complementary sequences has been determined to be interrupted by atruncated cross-arm sequence, wherein the permissive cell expresses arolling circle replication protein, but does not express viral capsidproteins capable of packaging replicative copies of the nucleic acidinto a viral particle; and (ii) maintaining the permissive cell underconditions in which the rolling circle replication protein in thepermissive cell replicates the nucleic acid.
 55. The method of claim 54,further comprising isolating the replicated nucleic acid from thepermissive cell.
 56. A method of analyzing a nucleic acid, the methodcomprising: i) obtaining a nucleic acid preparation comprising nucleicacid replication products isolated from a permissive cell, wherein thepermissive cell comprises a nucleic acid comprising a heterologousnucleic acid insert flanked by interrupted self-complementary sequences,each self-complementary sequence having an operative terminal resolutionsite and a rolling circle replication protein binding element, whereinone self-complementary sequence is interrupted by a cross-arm sequencethat forms two opposing, lengthwise-symmetric stem-loops, wherein theother of the self-complementary sequences is interrupted by a truncatedcross-arm sequence, wherein the permissive cell expresses a rollingcircle replication protein, but does not express viral capsid proteinscapable of packaging replicative copies of the nucleic acid into a viralparticle, and wherein the rolling circle replication protein binds tothe rolling circle replication protein binding element of the nucleicacid and replicates the nucleic acid to produce nucleic acid replicationproducts; and ii) determining a physiochemical property of one or morereplication products.
 57. The method of claim 56, wherein thephysiochemical property is the nucleotide sequence of one or eachself-complementary sequence.
 58. The method of claim 56, wherein thephysiochemical property is the extent of multimerization of one or morereplication products.
 59. The method of claim 56, wherein thephysiochemical property is the stoichiometry of monomeric and/ormultimeric forms of the replication product in the nucleic acidpreparation.
 60. The method of claim 56, wherein the physiochemicalproperty is the susceptibility of one or more replication products todigestion with a restriction endonuclease.
 61. The method of claim 56,wherein the physiochemical property is the polarity of monomers in adimeric form of the replication product, wherein the polarity ishead-to-head, head-to-tail or tail-to-tail.
 62. The method of claim 56,wherein the physiochemical property is the molecular weight of one ormore replication products or of a fragment of a replication product. 63.The method of claim 62, wherein the molecular weight is of a fragment ofthe one or more replication products that comprises one or eachself-complementary sequence.
 64. The method of claim 62 or 63, whereinthe molecular weight is determined based on electrophoretic mobility.65. The method of claim 62 or 63, wherein the molecular weight isdetermined based on mass spectroscopy.
 66. The method of claim 62,wherein the molecular weight is of a fragment of the one or morereplication products, and wherein prior to determining the molecularweight the fragment is amplified by a reaction comprising primerextension by a polymerase.
 67. The method of claim 62, wherein thereaction comprising primer extension is a polymerase chain reaction. 68.A method of preparing nucleic acids, the method comprising: (i)introducing into a permissive cell a nucleic acid comprising aheterologous nucleic acid insert flanked by interruptedself-complementary sequences, each self-complementary sequence having anoperative terminal resolution site and a rolling circle replicationprotein binding element, wherein one self-complementary sequence isinterrupted by a cross-arm sequence that forms two opposing,lengthwise-symmetric stem-loops, wherein the other of theself-complementary sequences is interrupted by a truncated cross-armsequence, wherein the permissive cell expresses a rolling circlereplication protein, but does not express viral capsid proteins capableof packaging replicative copies of the nucleic acid into a viralparticle; and (ii) maintaining the permissive cell under conditions inwhich the rolling circle replication protein in the permissive cellreplicates the nucleic acid.
 69. The method of claim 68, furthercomprising isolating the replicated nucleic acid from the permissivecell.